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WO2025213150A1 - Procédés, kits et systèmes de mesure de l'expression de psa et de psma et méthodes de traitement du cancer sur la base de ceux-ci - Google Patents

Procédés, kits et systèmes de mesure de l'expression de psa et de psma et méthodes de traitement du cancer sur la base de ceux-ci

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Publication number
WO2025213150A1
WO2025213150A1 PCT/US2025/023339 US2025023339W WO2025213150A1 WO 2025213150 A1 WO2025213150 A1 WO 2025213150A1 US 2025023339 W US2025023339 W US 2025023339W WO 2025213150 A1 WO2025213150 A1 WO 2025213150A1
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Prior art keywords
psma
subject
expression
psa
modifications
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Inventor
Matthew EATON
Anthony D'IPPOLITO
Jacob E. BERCHUCK
Rehan VERJEE
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Precede Biosciences Inc
Dana Farber Cancer Institute Inc
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Precede Biosciences Inc
Dana Farber Cancer Institute Inc
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Publication of WO2025213150A1 publication Critical patent/WO2025213150A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • PCa Prostate cancer
  • PSMA Prostate-Specific Membrane Antigen
  • PSMA-targeting therapeutics e.g., antibody-drug conjugates and radioimmune conjugates
  • PSMA expression level is commonly used as a criteria for determining patient eligibility for treatment.
  • PSA Prostate-specific antigen
  • KLK3 gamma-seminoprotein or kallikrein-3
  • PSA is a glycoprotein enzyme encoded in humans by the KLK3 gene.
  • PSA is present in small quantities in the serum of men with healthy prostates, but is often elevated in the presence of prostate cancer or other prostate disorders. As such, PSA can be used to screen for prostate cancer.
  • the present disclosure is based, at least in part, on the demonstration that PSMA expression level in a subject can be measured by detecting and quantifying the presence of histone modifications and/or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample, e.g., a plasma sample obtained or derived from a subject.
  • cfDNA cell-free DNA
  • the present disclosure demonstrates that PSMA expression level can be determined in cancer cells in a subject, including, e g., PSMA expression level in prostate cancer (e.g., mCRPC).
  • PSMA expression level determined by detecting histone modifications and/or DNA methylation at one or more genomic loci in cfDNA from a liquid biopsy sample can be used as a proxy for established biomarkers for, e.g., monitoring, characterizing, diagnosing, and prognosing disease and/or determining patient eligibility for certain therapeutics.
  • biomarkers e.g., monitoring, characterizing, diagnosing, and prognosing disease and/or determining patient eligibility for certain therapeutics.
  • technologies described herein can be used to predict PSMA PET measurements (e.g., PSMA PET SUVmean).
  • PSMA PET measurements predicted by technologies described herein have been shown to closely match actual PSMA PET measurements and also to be predictive of response to PSMA-targeted agents in patients.
  • PSA expression level in a subject can be measured or predicted by detecting and quantifying the presence of histone modifications and/or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample, e.g., a plasma sample obtained or derived from a subject.
  • cfDNA cell-free DNA
  • Technologies described herein for measuring or predicting PSA expression can provide certain advantages as compared to assays that measure PSA protein concentrations directly (e.g., via ELISA or an enzymatic assay).
  • technologies described herein can be combined with one or more additional technologies that comprise measuring epigenome modifications (e.g., technologies described herein that comprise measuring PSMA expressing or predicting PSMA PET signal), allowing for multiple epigenome measurements to obtained using a single sample (which, in some embodiments, can comprise a small volume of sample (e.g., 5 mb or less, or about 1 mL of plasma).
  • a single sample which, in some embodiments, can comprise a small volume of sample (e.g., 5 mb or less, or about 1 mL of plasma).
  • Use of single sample is advantageous as it allows for, e.g., a reduced number of sample processing steps (i.e., epigenome measurements only have to be collected once, and can be used to perform multiple analytes), and improved patient convenience.
  • the present disclosure encompasses methods that quantify the presence of histone modifications and/or DNA methylation, as well as methods that assess chromatin accessibility and/or binding of one or more transcription factors at one or more genomic loci instead of (or in addition to) histone modifications and/or DNA methylation.
  • Liquid biopsies are now widely utilized in clinical oncology to detect cancer recurrence and inform therapeutic decisions.
  • most commercially available cfDNA assays only detect genetic mutations and not all disease states have a characteristic mutation that can be used for detection.
  • Technologies that detect epigenetic modifications offer numerous benefits over assays that detect genetic mutations, including, e.g., allowing the detection of disease states that a characteristic mutation has not been identified for, and/or providing measurements that are more directly relevant to a biological characteristic of interest (e.g., detecting increased transcription activation of a gene, rather than a mutation that has been previously shown to be correlated with activation for some subjects).
  • the present disclosure provides tools to analyze multiple epigenomic features from patient plasma, including DNA methylation, chromatin accessibility, and histone modifications.
  • the present disclosure demonstrates that epigenomic cfDNA profiling can be used to detect PSMA and/or expression levels as well as characterize disease severity, prognose patients, evaluate patient eligibility for certain therapeutics, and inform methods of treatment.
  • prostate cancer e.g., mCRPC
  • cfDNA profiling would be immediately clinically actionable, as guidelines currently recommend that prostate cancer be monitored using imaging methods that assess PSMA expression in tumors and administering certain therapeutics to patients on the basis of PSMA expression level.
  • the present disclosure includes, among other things, technologies for determining PSMA expression level and for the detection, monitoring, and/or treatment of prostate cancer based on PSMA expression level.
  • the present disclosure relates to the measurement of histone modifications in a sample obtained or derived from a subject to detect and/or treat prostate cancer based on PSMA expression level.
  • the present disclosure includes, among other things, histone modification measurements in cell-free DNA (cfDNA) that are associated with PSMA expression level, and which in various embodiments are useful, e.g., for detecting, monitoring, selecting treatment for, and/or treating prostate cancer.
  • cfDNA cell-free DNA
  • the present disclosure includes, among other things, histone modification measurements in cfDNA that are associated with increased PSMA expression, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and/or treating prostate cancer (e.g., mCRPC).
  • histone modification measurements in cfDNA can be used to detect or determine responsiveness of prostate cancer (e.g., mCRPC) to a therapy.
  • histone modification measurements in cfDNA can be used to monitor or predict progression of prostate cancer (e.g., mCRPC).
  • histone modification measurements in cfDNA can be used to inform therapeutic selection for a subject with prostate cancer (e.g., determine an initial therapy, predict patients that are likely to respond to a given therapy, and/or determine when therapy should be changed for a subject).
  • histone modification measurements in cfDNA can be used as a complement to other diagnostic methods (e.g., imaging methods, histology methods, and/or symptom-based methods) for monitoring and/or treating prostate cancer (e g., performed concurrently and/or subsequent to other methods).
  • a method of described herein can be performed in combination with one or more diagnostic assays that do not comprise measuring one or more epigenome features.
  • a method described herein can be performed in combination with one or more diagnostic assays for prostate cancer that use PSA level, biopsy measurements, histology measurements, and/or medical imaging tests.
  • technologies described herein can be used to screen patients (e.g., identify patients for treatment, diagnosis, etc.) to identify patients that may benefit from being tested using one or more additional diagnostic assays.
  • technologies described herein can be performed in conjunction with one or more diagnostic assays that do not comprise measuring one or more epigenome features.
  • technologies described herein can be used for subjects that have been previously screened using one or more diagnostic assays that do not comprise measuring one or more epigenome features.
  • a method can be performed on a subject who has already been screened using one or more diagnostic assays for prostate cancer, e.g., one or more diagnostic assays described herein).
  • the present disclosure includes exemplary genomic loci whose epigenetic modification status is associated with PSMA expression level.
  • these genomic loci are or include one or more enhancers regions.
  • these genomic loci are or include one or more promoter regions.
  • the present disclosure includes, among other things, technologies for determining PSA expression level and for the detection, monitoring, and/or treatment of prostate cancer and/or the selection of subjects for further screening for prostate cancer based on PSA expression level.
  • the present disclosure relates to the measurement of histone modifications in a sample obtained or derived from a subject to detect and/or treat prostate cancer based on PSA expression level.
  • the present disclosure includes, among other things, histone modification measurements in cell-free DNA (cfDNA) that are associated with PSA expression level, and which in various embodiments are useful, e.g., for detecting, monitoring, selecting treatment for, and/or treating prostate cancer.
  • cfDNA cell-free DNA
  • histone modification measurements in cfDNA that are associated with increased PSA expression, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and/or treating prostate cancer (e.g., mCRPC) or for identifying subjects for further screening for prostate cancer.
  • histone modification measurements in cfDNA can be used to detect or determine responsiveness of prostate cancer (e.g., mCRPC) to a therapy.
  • histone modification measurements in cfDNA can be used to monitor or predict progression of prostate cancer (e.g., mCRPC).
  • histone modification measurements in cfDNA can be used to inform therapeutic selection for a subject with prostate cancer (e.g., determine an initial therapy, predict patients that are likely to respond to a given therapy, and/or determine when therapy should be changed for a subject).
  • histone modification measurements in cfDNA can be used as a complement to other diagnostic methods (e.g., imaging methods and/or symptom-based methods) for detecting, monitoring, and/or treating prostate cancer (e.g., performed concurrently and/or subsequent to other methods).
  • the present disclosure includes exemplary genomic loci whose epigenetic modification status is associated with PSA expression level.
  • these genomic loci are or include one or more enhancer regions.
  • these genomic loci are or include one or more promoter regions.
  • a genomic locus is differentially modified if it is characterized by increased or decreased histone modification as compared to a reference (e.g., a sample from a healthy subject).
  • Increased or decreased histone modification can be or include, e.g., increased or decreased histone methylation (hypermethylation or hypomethylation, respectively) of one or more particular methylation marks, or a combination thereof; increased or decreased pan-methylation; increased or decreased histone acetylation (hyperacetylation or hypoacetylation, respectively) of one or more particular acetylation marks, or a combination thereof; and/or increased or decreased pan-acetylation (e.g., pan-H3 acetylation).
  • the present disclosure further relates, in various embodiments, to the measurement of chromatin accessibility in cell-free DNA (cfDNA) to determine PSMA expression level.
  • cfDNA cell-free DNA
  • the present disclosure includes, among other things, chromatin accessibility measurements in cfDNA that are characteristic of increased PSMA expression in a tumor, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and/or treating prostate cancer.
  • chromatin accessibility measurements in cfDNA that are characteristic of increased PSMA expression, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and/or treating prostate cancer.
  • the present disclosure further relates, in various embodiments, to the measurement of chromatin accessibility in cell-free DNA (cfDNA) to determine PSA expression level.
  • cfDNA cell-free DNA
  • the present disclosure includes, among other things, chromatin accessibility measurements in cfDNA that are characteristic of increased PSA expression in a tumor, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, treating, and/or screening for prostate cancer.
  • chromatin accessibility measurements in cfDNA that are characteristic of increased PSA expression, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, treating, and/or screening for prostate cancer.
  • chromatin accessibility measurements in cfDNA can be used to detect or determine resistance of prostate cancer to a therapy.
  • the present disclosure includes genomic loci that are differentially accessible when PSA expression is increased.
  • genomic loci differentially accessible in cfDNA are or include one or more enhancers.
  • genomic loci differentially accessible in cfDNA are or include one or more promoters.
  • histone methylation e.g., H3K4me3 corresponds and/or is correlated with chromatin accessibility.
  • histone acetylation corresponds and/or is correlated with chromatin accessibility.
  • DNA methylation corresponds and/or is correlated with chromatin accessibility.
  • the present disclosure further relates, in various embodiments, to the measurement of transcription factor binding in cell-free DNA (cfDNA) to determine PSMA expression level.
  • the present disclosure includes, among other things, transcription factor binding measurements in cfDNA that are characteristic of increased PSMA expression, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and/or treating prostate cancer.
  • transcription factor binding measurements in cfDNA can be used to detect or determine resistance of prostate cancer to a therapy.
  • the present disclosure includes genomic loci that are differentially bound by transcription factors when PSMA expression is increased.
  • genomic loci that are differentially bound by transcription factors in cfDNA are or include one or more enhancers.
  • genomic loci that are differentially bound by transcription factors in cfDNA are or include one or more promoters.
  • the present disclosure further relates, in various embodiments, to the measurement of transcription factor binding in cell-free DNA (cfDNA) to determine PSA expression level.
  • the present disclosure includes, among other things, transcription factor binding measurements in cfDNA that are characteristic of increased PSA expression, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, screening for, and/or treating prostate cancer.
  • transcription factor binding measurements in cfDNA can be used to detect or determine resistance of prostate cancer to a therapy.
  • the present disclosure includes genomic loci that are differentially bound by transcription factors when PSA expression is increased.
  • genomic loci that are differentially bound by transcription factors in cfDNA are or include one or more enhancers.
  • genomic loci that are differentially bound by transcription factors in cfDNA are or include one or more promoters.
  • histone methylation corresponds and/or is correlated with transcription factor binding.
  • histone acetylation corresponds and/or is correlated with transcription factor binding.
  • DNA methylation corresponds and/or is correlated with transcription factor binding.
  • a genomic locus is differentially bound by transcription factors if it is characterized by increased or decreased transcription factor binding as compared to a reference (e.g., a sample from a healthy subject).
  • Increased or decreased transcription factor binding can be or include, e.g., increased or decreased transcription factor binding as determined by various transcription factor binding assays known in the art.
  • the present disclosure provides a method of determining PSMA expression level in a subject, the method comprising: quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) binding of one or more transcription factors, and/or (iv) DNA methylation.
  • cfDNA cell-free DNA
  • the one or more histone modifications are quantified using a histone modification assay that measures one or more of H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4mel, H3K4me2, H3K4me3, and pan-acetylation.
  • the histone modification assay detects H3K4me3 modifications.
  • the histone modification assay detects H3K27ac modifications.
  • the histone modification assay is selected from ChlP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.
  • chromatin accessibility is quantified using a chromatin accessibility assay selected from ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde- Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), a Dnase hypersensitivity assay, and a fragmentomics assay.
  • ATAC-seq Assay of Transpose Accessible Chromatin sequencing
  • NOMe-seq Nucleosome Occupancy and Methylome sequencing
  • FAIRE-seq Formmaldehyde- Assisted Isolation of Regulatory Elements sequencing
  • MNase-seq Merococcal Nuclease digestion with sequencing
  • Dnase hypersensitivity assay and a fragmentomics assay.
  • binding of one or more transcription factors is quantified using a transcription factor binding assay.
  • the transcription factor binding assay is selected from ChlP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.
  • DNA methylation is quantified using Bisulfite sequencing (BS-Seq), Whole Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or Methyl-CpG-Binding Domain sequencing (MBD-seq).
  • BS-Seq Bisulfite sequencing
  • WGBS Whole Genome Bisulfite Sequencing
  • MBD-seq Methyl-CpG-Binding Domain sequencing
  • a method comprises quantifying two or more of the following, each at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) transcription factor binding, and/or (iv) DNA methylation.
  • the method comprises quantifying two or more histone modifications, e.g., quantifying H3K4me3 and H3K27ac modifications.
  • a method comprises quantifying one or more histone modifications and DNA methylation, e.g., quantifying H3K4me3 and/or H3K27ac modifications and DNA methylation.
  • a method comprises quantifying H3K4me3 modifications, H3K27ac modifications and DNA methylation.
  • a biological sample is a liquid biopsy sample, e.g., a plasma sample, serum sample, or urine sample.
  • quantification of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at the one or more genomic loci as compared to a reference indicates that the subject has increased PSMA expression.
  • the reference is a predetermined threshold, a measurement from a liquid biopsy sample, and/or a normalized value, optionally wherein the reference is a measurement from a liquid biopsy sample obtained from a cohort of healthy subjects.
  • technologies described herein comprise or can be used to measure PSMA expression and/or predict medical imaging results (e.g., PSMA PET imaging results).
  • a method for measuring PSMA expression measures PSMA expression specific to one or more tumors in a subject.
  • PSMA expression comprises cell surface expression.
  • PSMA expression comprises tumor cell specific expression.
  • PSMA expression comprises tumor specific, cell surface expression of PSMA.
  • one or more histone modifications are quantified using a histone modification assay that measures H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4mel, H3K4me2, H3K4me3, or pan-acetylation, or any combination thereof.
  • a histone modification assay detects H3K4me3 modifications.
  • a histone modification assay detects H3K27ac modifications.
  • chromatin accessibility is quantified using a chromatin accessibility assay selected from ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing), Mnase-seq (Micrococcal Nuclease digestion with sequencing), a Dnase hypersensitivity assay, and a fragmentomics assay.
  • ATAC-seq Assay of Transpose Accessible Chromatin sequencing
  • NOMe-seq Nucleosome Occupancy and Methylome sequencing
  • FAIRE-seq Formmaldehyde-Assisted Isolation of Regulatory Elements sequencing
  • Mnase-seq Merococcal Nuclease digestion with sequencing
  • Dnase hypersensitivity assay and a fragmentomics assay.
  • binding of one or more transcription factors
  • a transcription factor binding assay is selected from ChlP- seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.
  • a method described herein comprises quantifying two or more histone modifications. In some embodiments, a method described herein comprises quantifying H3K4me3 and H3K27ac modifications. In some embodiments, a method described herein comprises quantifying one or more histone modifications and DNA methylation. In some embodiments, a method described herein comprises quantifying H3K4me3 and/or H3K27ac modifications and DNA methylation. In some embodiments, a method described herein comprises quantifying H3K4me3 modifications, H3K27ac modifications, and DNA methylation. [0050] In some embodiments, a liquid biopsy sample is a plasma sample, serum sample, or urine sample.
  • an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci as compared to a reference indicates that a subject has increased PSMA expression (e.g., increased as compared to a healthy subject).
  • a method described herein comprises measuring one or more prostate cancer specific markers.
  • one or more prostate cancer specific markers comprise PSA expression (e.g., PSA serum level).
  • PSA expression is measured by measuring PSA protein concentrations (e.g., via an ELISA assay and/or an enzymatic assay).
  • PSA expression is measured by quantifying histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci (e.g., using technologies described in the present disclosure).
  • a method comprises measuring PSMA expression or predicting PSMA expression (e.g., predicting a PSMA PET measurement) and measuring one or more prostate cancer specific markers.
  • a method described herein comprises quantifying one or more histone modifications and/or DNA methylation at one or more prostate cancer specific marker genes or regulatory regions thereof (e.g., one or more promoters and/or enhancers of one or more prostate cancer specific marker genes).
  • one or more prostate cancer specific marker genes comprise HXBI3, KLK2, KLK3, SPDEF, or FOLH1, or any combination thereof.
  • a method described herein comprises quantifying one or more histone modifications and/or DNA methylation for one or more of AMN, ARHGEF37, Clorf 36, CADM1, CCDC175, CDC7, CLSTN1, COL5A1, EDNRA, FOLH1, GALR3, MED13L, MICB, NDRG3, NEDD1, NPAS2, NPVF, OLFM1, PCBP4, PROZ, PRRG3, RREB1, SCUBE3, SERPINA5, SNRPF, SORCS3, ST8SIA5, TEX19, TMEM132B, or TTC29 or any combination thereof, or one or more regulatory regions of any one of the foregoing (e.g., one or more promoter and/or enhancer regions oiAMN, ARHGEF37, C4orf36, CADM1, CCDC175, CDC7, CLSTNI, COL5A1, EDNRA, FOLH1, GALR3, MED13L, MICB, NDRG3,
  • genomic loci that are associated with PSMA expression and can be used to measure PSMA expression (e.g., tumor specific PSMA expression).
  • Exemplary genomic loci include those provided in Tables 1 and 2.
  • a method described herein comprises quantifying:
  • promoter signal e.g., H3K4me3
  • H3K4me3 promoter signal
  • CADM1 CDC7
  • COL5A1 promoter region of C4orf36
  • EDNRA e.g., CDC7
  • COL5A1 e.g., EDNRA
  • MED13L e.g., ED13L
  • PROZ e.g., SNRPF, TEX19, or any combination thereof
  • enhancer signal e.g., H3K27ac signal
  • enhancer signal at one or more enhancer regions of ARHGEF37, CLSTNI, FOLH1, NDRG3, NPAS2, NPVF, 0LFM1, RREBl, SCUBE3, or TTC29, or any combination thereof;
  • a method described herein comprises:
  • a method described herein comprises:
  • promoter signal e.g., H3K4me3 modifications
  • a method described herein comprises quantifying:
  • promoter signal e.g., H3K4me3 modifications
  • H3K4me3 modifications for 1 or 2 H3K4me3 analyte genomic loci in Table 4;
  • enhancer signal e.g., H3K27ac modifications
  • H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 H3K27ac analyte genomic loci in Table 4;
  • a method described herein comprises quantifying (a) promoter signal (e.g., H3K4me3 modifications) at chrl 1 :49,228,902-49,230,855; and/or (b) enhancer signal (e.g., H3K27ac modifications) at chrl 1 :49, 229, 019-49, 230, 275.
  • promoter signal e.g., H3K4me3 modifications
  • enhancer signal e.g., H3K27ac modifications
  • promoter signal and enhancer signal are normalized prior to aggregating (e.g., normalized based on sequence read depth or ctDNA fraction).
  • aggregating comprises summing epigenetic modification signal (e.g., H3K4me3 and/or H3K27ac signal) at two or more loci.
  • signal is corrected prior to summing.
  • correcting comprises adjusting for (i) sequencing depth, (ii) background signal (e.g., signal in healthy subjects), (iii) the length of a genomic locus, or any combination of (i)-(iii).
  • signal (optionally corrected signal) at each locus is weighted when aggregated.
  • weighting entails multiplying by a coefficient that has been determined using a model trained to predict PSA or PSMA expression.
  • promoter signal and enhancer signal are separately aggregated, and then the aggregated promoter signal and enhancer signal are aggregated in a second aggregation step. In some embodiments, promoter signal and enhancer signal are aggregated together (i.e., without an intervening aggregation step).
  • a biological sample e.g., a plasma sample
  • a subject has previously been diagnosed with a disease or condition that is associated with increased PSMA expression.
  • a disease or condition that is associated with increased PSMA expression is cancer.
  • a disease or indication associated with PSMA expression is prostate cancer.
  • prostate cancer is mCRPC (metastatic castration resistant prostate cancer).
  • prostate cancer is prostate adenocarcinoma (PRAD).
  • prostate cancer is neuroendocrine prostate cancer (NEPC).
  • a subject has previously been diagnosed with a disease or condition that is associated with increased PSA expression.
  • a disease or condition that is associated with increased PSA expression is cancer.
  • a disease or indication associated with PSA expression is prostate cancer.
  • prostate cancer is mCRPC (metastatic castration resistant prostate cancer).
  • prostate cancer is prostate adenocarcinoma (PRAD).
  • prostate cancer is neuroendocrine prostate cancer (NEPC).
  • one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and DNA/or DNA methylation is quantified in a subject before the subject is administered a PSMA-targeted agent.
  • the present disclosure describes a method of predicting PSA expression in a subject, the method comprising: quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA) from a liquid biopsy sample, obtained or derived from the subject:
  • cfDNA cell-free DNA
  • ctDNA circulating tumor DNA
  • PSA expression is or comprises serum PSA concentration.
  • a method measures or predicts PSA expressed by one or more cancer cells in the subject.
  • a method measures or predicts serum concentration of total PSA. [0076] In some embodiments, a method predicts PSA expression as determined using an assay that (a) utilizes one or more antibodies that bind PSA (e.g., an ELISA assay) and/or (b) measures PSA enzymatic activity.
  • an assay that (a) utilizes one or more antibodies that bind PSA (e.g., an ELISA assay) and/or (b) measures PSA enzymatic activity.
  • a method of predicting a likelihood that a subject having a disease or disorder associated with increased PSA expression will respond to a therapeutic comprises measuring PSA expression or predicting PSA expression in a subject using a method described herein, and comparing the measured PSA expression level or predicted PSA expression to a reference, wherein
  • a method of treating a subject having a disease or disorder associated with increased PSA expression comprises measuring or predicting PSA expression (e.g., serum PSA, including, e.g., total serum PSA) in a subject using a method described herein.
  • a method of treating a subject having a disease or disorder associated with increased PSA expression comprises measuring or predicting PSA expression (e.g., serum PSA, including, e.g., total serum PSA) in a subject using a method described herein, and comparing the measured PSA or predicted PSA expression to a reference, and
  • a reference is the median, lower bound of the top tertile, or lower bound of the top quartile value of a PSMA expression level measured, tumor specific PSMA expression level (e.g., PSMA PET SUVmean value) predicted, or tumor specific PSMA expression level (e.g., PSMA PET SUVmean value) measured in a cohort of subjects having a disease or disorder associated with increased PSMA expression (e.g., prostate cancer, including, e g., mCRPC); and if the PSMA expression level or predicted tumor specific PSMA expression level in the subject is equal to or greater than the reference, administering a PSMA targeted therapeutic (e.g., 177Lu-PSMA-617); and if the PSMA expression level or predicted tumor specific PSMA expression level in the subject is less than the reference, not administering the PSMA targeted therapeutic.
  • a PSMA targeted therapeutic e.g., 177Lu-PSMA-617
  • the PSMA PET SUVmean median is about 4 to about 8, about 5 to about 8, 5 to about 7, about 6 to about 9, about 5, about 6, about 7, or about 8 in a reference population.
  • the lower bound of the top tertile of PSMA PET SUVmean is about 6 to about 12, about 6 to about 10, about 8 to about 12, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 in a reference population.
  • the lower bound of the top quartile of PSMA PET SUVmean is about 6 to about 14, about 6 to about 12, about 8 to about 14, about 8, about 9, about 10, about 11, about 12, about 13, or about 14 in a reference population.
  • a disease or indication associated with increased PSMA expression is a cancer.
  • the cancer is prostate cancer, including, e.g., mCRPC.
  • a therapeutic is administered via one or more intravenous, subcutaneous, intraperitoneal, or intramuscular injections.
  • a subject has previously been diagnosed as having a disease or indication associated with increased PSMA or PSA expression (e.g., a cancer, prostate cancer, mCRPC, PRAD and/or NEPC).
  • a disease or indication associated with increased PSMA or PSA expression e.g., a cancer, prostate cancer, mCRPC, PRAD and/or NEPC.
  • a prostate cancer is localized or metastatic.
  • a prostate cancer has metastasized to one or more site(s) that include lymph node, bone, lung, and/or liver tissue.
  • a subject has previously been administered one or more
  • promoter signal e.g., H3K4me3 modifications
  • H3K4me3 modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 H3K4me3 analyte genomic loci in Table 1;
  • enhancer signal e.g., H3K27ac modifications
  • H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 H3K27ac analyte genomic loci in Table 1;
  • promoter signal e.g., H3K4me3 modifications
  • kits comprises one or more antibodies for use in ChlP- seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac- modified histones.
  • a kit comprises one or more methyl-binding domains for use in MBD-seq or wherein the kit comprises one or more antibodies that bind methylated DNA for use in MeDIP.
  • a kit comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample.
  • a kit comprises reagents for library preparation for sequencing.
  • a kit comprises reagents for sequencing.
  • described herein is a non-transitory computer readable storage medium encoded with a computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform a method described herein.
  • a computer system comprising a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform operations to perform a method described herein.
  • a sequencer is configured to generate a Whole Genome Sequencing (WGS) data set from the sample.
  • WGS Whole Genome Sequencing
  • a system comprises a sample preparation device configured to prepare a sample for sequencing from a biological sample, optionally a liquid biopsy sample.
  • promoter signal e.g., H3K4me3 modifications
  • H3K4me3 modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 H3K4me3 analyte genomic loci in Table 1;
  • enhancer signal e.g., H3K27ac modifications
  • H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 H3K27ac analyte genomic loci in Table 1;
  • enhancer signal e.g., H3K27ac modifications
  • promoter signal e.g., H3K4me3 modifications
  • enhancer signal e.g., H3K27ac modifications
  • promoter signal e.g., H3K4me3 modifications
  • promoter signal e.g., H3K4me3 modifications
  • enhancer signal e.g., H3K27ac modifications
  • a system described herein comprises reagents that comprise one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones.
  • a device comprises reagents for isolation of cell-free DNA (cfDNA) from a biological sample, optionally a liquid biopsy sample.
  • cfDNA cell-free DNA
  • a system comprises a device that comprises reagents for library preparation for sequencing.
  • a sequencer comprises reagents for sequencing.
  • the present disclosure is based, at least in part, on the demonstration that certain genomic loci associated with an ADC target antigen (FOLH1, encoding the ADC target antigen PSMA) have different histone modification levels (e.g., histone methylation marks such as H3K4me3 and histone acetylation marks such as H3K27ac) in plasma samples from cancer patients (e.g., prostate cancer) as compared to plasma samples from healthy volunteers.
  • FOLH1 histone methylation marks
  • H3K27ac histone acetylation marks
  • the present disclosure encompasses methods, kits and systems that use epigenomic differences (alone or in combination with each other and/or with other biomarkers) to select subjects for treatment with an agent that is directed to FOLH1 (e.g., an ADC therapy or radioligand directed to PSMA), to identify subpopulations of subjects that respond to treatment with an agent that is directed to FOLH1, to monitor subjects during treatment with an agent that is directed to FOLHl (e.g., an ADC therapy or radioligand directed to PSMA), etc. by detecting and quantifying the presence of histone modifications at these one or more genomic loci in cell- free DNA (cfDNA) from a liquid biopsy sample, e.g., a plasma sample obtained or derived from a subject.
  • cfDNA cell- free DNA
  • the present disclosure also encompasses methods where chromatin accessibility and/or binding of one or more transcription factors are detected at the one or more genomic loci instead of (or in addition to) histone modifications.
  • the present disclosure also encompasses methods, kits and systems where the genomic loci that are differentially modified based on different types of histone modifications (e.g, histone methylation marks such as H3K4me3 and histone acetylation marks such as H3K27ac) are combined into multimodal classifiers to select subjects for treatment with an agent that is directed to F0LH1 (e.g., an ADC therapy or radioligand directed to PSMA), etc.
  • F0LH1 e.g., an ADC therapy or radioligand directed to PSMA
  • These monomodal and multimodal classifiers can provide minimally invasive ways of selecting subjects for treatment with an agent that is directed to F0LH1 (e.g., an ADC therapy or radioligand directed to PSMA), etc. that are more accurate, objective, and comprehensive than the current tissue-based approaches.
  • an agent that is directed to F0LH1 e.g., an ADC therapy or radioligand directed to PSMA
  • the present disclosure includes, among other things, technologies for the determination of the activation status of F0LH1 and for the detection, monitoring, and/or treatment of cancer (including, e.g., prostate cancer, breast cancer, SCLC, NSCLC, etc.) based on the activation status of these genes.
  • cancer including, e.g., prostate cancer, breast cancer, SCLC, NSCLC, etc.
  • the present disclosure relates to the measurement of histone modifications in a sample obtained or derived from a subject to detect and/or treat cancer (including, e.g., prostate cancer, breast cancer, SCLC, NSCLC, etc.) based on the activation status of these genes.
  • the present disclosure includes, among other things, histone modification measurements in cell-free DNA (cfDNA) that are characteristic of the activation status of genes for F0LH1, and which in various embodiments are useful, e.g., for detecting, monitoring, selecting treatment for, and/or treating cancer (including, e.g., prostate cancer, breast cancer, SCLC, NSCLC, etc.) based on the activation status of these genes.
  • cfDNA cell-free DNA
  • histone modification measurements in cfDNA can be used to detect or determine resistance of a cancer (e.g., prostate cancer, breast cancer, SCLC, NSCLC, etc.) to treatment with an agent that is directed to FOLH1 (e.g., an ADC therapy or radioligand directed to PSMA) or transformation of a cancer from one subtype to another.
  • the present disclosure includes exemplary genomic loci that are differentially modified in different cancer patients (including, e.g., prostate cancer, breast cancer, SCLC, NSCLC, etc. patients) and/or between cancer patients and healthy volunteers.
  • genomic loci differentially modified in cfDNA are or include one or more enhancers.
  • genomic loci differentially modified in cfDNA are or include one or more promoters.
  • a genomic locus is differentially modified if it is characterized by increased or decreased histone modification as compared to a reference (e.g., a sample from a PSMA-negative or healthy subject).
  • Increased or decreased histone modification can be or include, e.g., increased or decreased histone methylation (hypermethylation or hypomethylation, respectively) of one or more particular methylation marks, or a combination thereof; increased or decreased pan-methylation; increased or decreased histone acetylation (hyperacetylation or hypoacetylation, respectively) of one or more particular acetylation marks, or a combination thereof; and/or increased or decreased pan-acetylation (e.g., pan-H3 acetylation).
  • histone methylation can be or include histone methylation marks selected from H3K4mel, H3K4me2, H3K4me3, or a combination thereof. In various embodiments, histone methylation can be or include H3K4me3. In various embodiments, histone acetylation can be or include histone acetylation marks selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, or a combination thereof. In various embodiments, histone acetylation can be or include H3K27ac.
  • the present disclosure further relates, in various embodiments, to the measurement of chromatin accessibility in cell-free DNA (cfDNA) to determine the activation status of FOLH1.
  • cfDNA cell-free DNA
  • the present disclosure includes, among other things, chromatin accessibility measurements in cfDNA that are characteristic of PSMA-positive cancers, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and/or treating a PSMA-positive cancer.
  • histone acetylation corresponds and/or is correlated with chromatin accessibility.
  • histone methylation corresponds and/or is correlated with chromatin accessibility.
  • a genomic locus is differentially accessible if it is characterized by increased or decreased chromatin accessibility as compared to a reference (e.g, a sample from an ADC target-negative or healthy subject).
  • Increased or decreased histone modification can be or include, e.g., increased or decreased accessibility as determined by various chromatin accessibility assays known in the art.
  • the present disclosure includes genomic loci that are differentially bound by transcription factors in PSMA-positive vs. PSMA- negative cancers.
  • genomic loci that are differentially bound by transcription factors in cfDNA are or include one or more enhancers.
  • genomic loci that are differentially bound by transcription factors in cfDNA are or include one or more promoters.
  • the present disclosure provides a method comprising quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from a subject: (i) one or more histone modifications, (ii) DNA methylation, (iii) chromatin accessibility, and/or (iv) binding of one or more transcription factors, wherein the one or more genomic loci are (a) within a gene encoding PSMA.
  • cfDNA cell-free DNA
  • a method comprises quantifying enhancer signal (e.g., H3K27ac modifications) at one or more loci within a certain distance of KLK3 (e.g., within 500 kb, 400 kb, 300 kb, 200 kb, 150 kb, 100 kb, or 50 kB of KLK3).
  • enhancer signal e.g., H3K27ac modifications
  • a method comprises quantifying promoter signal (e.g., H3K4me3) at one or more loci within a certain distance of KLK3 (e g., 500 kb, 400 kb, 300 kb, 200 kb, 150 kb, 100 kb, 50 kB, 20 kB, 10 kB, 5, kB, 4 kB, 3 kB, 2 kB, or 1 kB of KLK3).
  • promoter signal e.g., H3K4me3
  • KLK3 e.g., 500 kb, 400 kb, 300 kb, 200 kb, 150 kb, 100 kb, 50 kB, 20 kB, 10 kB, 5, kB, 4 kB, 3 kB, 2 kB, or 1 kB of KLK3
  • a method comprises quantifying DNA methylation at one or more loci within the transcript encoding portion of KLK3 and/or at one or more loci within a certain distance of KLK3 (e.g., within 500 kb, 400 kb, 300 kb, 200 kb, 150 kb, 100 kb, or 50 kB of KLK3).
  • one or more loci within a certain distance of KLK3 include one or more loci with differential H3K4me3, H3K27ac, and/or DNA methylation signal (e.g., as compared to a healthy subject).
  • one or more loci within a certain distance of KLK3 include one or more loci at which levels of H3K4me3, H3K27ac, and/or DNA methylation signal is correlated with PSA expression (e.g., as determined by quantifying RNA transcript (e.g., as determined using an RNA-seq assay using tumor samples, PDX samples, and/or one or more cell lines).
  • a method comprises quantifying enhancer signal (e.g., H3K27ac modifications) at one or more loci within a certain distance of FOLH1 (e.g., within 500 kb, 400 kb, 300 kb, 200 kb, 150 kb, 100 kb, or 50 kB o FOLHl .
  • enhancer signal e.g., H3K27ac modifications
  • a method comprises quantifying promoter signal (e.g., H3K4me3) at one or more loci within a certain distance oiFOLHl (e.g., 500 kb, 400 kb, 300 kb, 200 kb, 150 kb, 100 kb, 50 kB, 20 kB, 10 kB, 5, kB, 4 kB, 3 kB, 2 kB, or 1 kB oiFOLHl .
  • promoter signal e.g., H3K4me3
  • oiFOLHl e.g., 500 kb, 400 kb, 300 kb, 200 kb, 150 kb, 100 kb, 50 kB, 20 kB, 10 kB, 5, kB, 4 kB, 3 kB, 2 kB, or 1 kB oiFOLHl .
  • one or more loci within a certain distance of FOLH1 include one or more loci with differential H3K4me3, H3K27ac, and/or DNA methylation signal (e g., as compared to a healthy subject).
  • one or more loci within a certain distance of FOLH1 include one or more loci at which levels of H3K4me3, H3K27ac, and/or DNA methylation signal is correlated with PSMA expression (e.g., as determined by quantifying RNA transcript (e.g., as determined using an RNA-seq assay using tumor samples, PDX samples, and/or one or more cell lines).
  • Fig. 1 is a schematic showing a summary of an exemplary comprehensive epigenomic platform offering dynamic resolution into target and pathway biology from plasma.
  • A Cell free DNA derived from tumors exists in circulation as chromatin fragments that faithfully maintain tumor-associated epigenetic modifications on histones and DNA. Antibodies against H3K27ac (marking active enhancers), H3K4me3 (marking active promoters), and DNA methylation can be used to enrich for associated DNA fragments from plasma, which can then be sequenced to provide genome-wide epigenomic maps that capture the underlying transcriptional state of tumor cells.
  • B Clinical study overview of mCRPC patient plasma samples evaluated in the study described in Example 2.
  • Fig. 2 shows volcano plots demonstrating identification of plasma epigenomic features that associate with mCRPC and PSMA-PET signal.
  • “Up’7 “Down” labels indicate the number of statistically-significant loci for enhancers, promoters, and DNA methylation that are upregulated (Up) or downregulated (Down) in mCRPC patients vs healthy volunteers. Labels represent the nearest TSS to statistically significant peaks of interest.
  • the x-axis values represent the slope of the association between the z-score of the epigenomic feature and the z-score of PSMA PET SUVmean, and the y-axis is the statistical significance of that association.
  • the top three features for each analyte are labeled with their closest gene (TSS).
  • Fig. 3 shows exemplary enrichment tracks demonstrating that the F0LH1 locus has robust enhancer and promoter signal in mCRPC patients compared to healthy volunteers.
  • Enhancer, promoter, and DNA methylation signal in mCRPC patients with either high or low PSMA PET SUV mean WHS normalized, smoothed, and averaged together (within analyte) with the mean signal from a cohort of male healthy volunteers.
  • Fig. 4 shows exemplary enrichment tracks demonstrating that the FOLH1 locus has higher enhancer and promoter signal in patients with higher PSMA PET SUVmean.
  • Enhancer, promoter, and DNA methylation signal in mCRPC patients with either high or low PSMA PET SUV mean W3S normalized, smoothed, and averaged together (within analyte) with the mean signal from a cohort of male healthy volunteers.
  • FIG. 5 shows scatter plots demonstrating that enhancer and promoter signal at the FOLH1 locus predicts PSMA PET SUVmean in both cross-validation and in validation cohort.
  • Enhancer and promoter signal at the FOLH1 locus were used to train a machine learning (ML) model to predict PSMA PET SUVmean.
  • Samples were first split into training and validation cohorts, which were matched for ctDNA% and PSMA PET SUVmean distributions.
  • training cohort samples (ctDNA% >3) were used to identify robust, mCRPC-specific enhancer/promoter regions at the FOLH1 locus. Signal at these regions were then used train a model to predict the corresponding PSMA PET SUVmean quantifications. Performance was assessed via Pearson correlation in both a leave-one-out (LOO) cross-validation (CV) setting within the training cohort, as well as the held-out validation cohort using a final model trained on all data from the training cohort.
  • LEO leave-one-out
  • CV cross-validation
  • FIG. 6 shows survival graphs demonstrating association with clinical outcomes with PSMA PET scores predicted using a model described herein. Shown are clinical outcomes (as measured by 4 clinical trial metrics) for mCRPC subjects having different PSMA PETtreated with lutetium-177 (177Lu)-PSMA-617, having different PSMA scores as determined using methods provided herein.
  • A Shows progression free survival as determined by whether there is an increase in serum PSA (PSA-PFS).
  • B Shows “crPFS” values, referring to progression free survival based on clinical or radiological evidence of progression.
  • C Shows Time to Next Treatment.
  • D Shows overall survival. In each of (A)-(D), lines represent patients with a PSMA score, from left to right, in the bottom tertile of patients, in the middle tertile of patients, in the top tertile of patients.
  • FIG. 7 shows survival graphs for comparison of hazard ratios based on PSMA PET SUVmean predictions vs. PSMA PET measured values. Shown is a comparison of clinical trial outcomes for a cohort of mCRPC patients administered lutetium-177 (177Lu)-PSMA-617, and having different measured PSMA PET SUVmean values (PSMA PET SUVmean (Measured)) vs. predicted PSMA PET SUVmean values (PSMA PET SUVmean (Predicted)), which were predicted using technologies described herein.
  • (A) and (B) show progression free survival as determined by whether an increase in serum PSA was observed (PSA-PFS), in subjects having different measured PSMA PET SUVmean or predicted PSMA PET SUVmean values, respectively.
  • (C) and (D) show Time to Next Treatment (TTNT), in subjects having different measured PSMA PET SUVmean or predicted PSMA PET SUVmean values, respectively.
  • (E) and (F) show “crPFS” values, referring to progression free survival based on clinical or radiological evidence of progression, in subjects having different measured PSMA PET SUVmean or predicted PSMA PET SUVmean values, respectively.
  • (G) and (H) show Overall Survival (OS), in subjects having different measured PSMA PET SUV mean or predicted PSMA PET SUVmean values, respectively.
  • black lines represent patients with a measured or predicted PSMA PET SUVmean in the top tertile of patients and grey lines represent patients with a measured or predicted PSMA PET SUVmean in the middle and bottom tertiles of patients.
  • HR refers to Hazard Ratio.
  • Fig. 8 shows a scatterplot with comparison of predicted and measured PSA expression in plasma samples from prostate cancer patients. Shown is predicted PSA expression (“Predicted KLK3 RNA-seq expression,” y-axis), determined using epigenetic modification measurements collected in plasma samples obtained from prostate cancer patients, and serum PSA (“PSA plasma concentration,” x-axis) measured in matched plasma samples, p refers to Pearson’s coefficient. Shading indicates 95% confidence interval.
  • FIG. 9 shows a scatterplot demonstrating prediction of PSMA PET SUVmean using patient plasma samples.
  • A Shows PSMA expression predicted using a biopsy model, using epigenetic modification measurements from plasma samples obtained from prostate cancer patients (y-axis) vs. PSMA PET SUVmean measured in matched patients (x-axis).
  • B Shows PSMA PET SUVmean predicted using a model generated using patient plasma data and PMSA PET SUVmean values vs. PSMA PET SUVmean measured in matched patients (x-axis).
  • Fig. 11 shows clinicoradiographic Progression Free Survival (CR PFS) for mCRPC patients with a predicted PSMA PET SUVmean value in the top tertile (top, black line) and middle and bottom tertiles (red, bottom line).
  • HR refers to Hazard Ratio.
  • Figs. 12(A)-(D) show prostate specific antigen (PSA), time to next treatment (TTNT), clinicoradiographic Progression Free Survival (CR PFS), and overall survival (OS) metrics for patients with mCRPC with ctDNA% in the top tertile (bottom, black line) and middle and bottom tertiles (red, top line).
  • HR refers to Hazard Ratio.
  • PSMA expression level e.g., PSMA expression level
  • a subject can be determined by detecting and quantifying the presence of histone modifications and/or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample, e.g., a plasma sample obtained or derived from a subject.
  • cfDNA cell-free DNA
  • the present disclosure demonstrates that PSMA expression level can be determined in cancer cells in a subject, including, e.g., PSMA expression level in prostate cancer (e.g., mCRPC).
  • Determining PSMA expression level can be used for, e.g., diagnosing, prognosing, or monitoring a disease in a subject, and methods of treatment (e.g., for identifying subject more likely to respond to treatment with a PSMA-targeted therapeutic).
  • Prostate cancer is a disease characterized by the uncontrolled growth of cells in the prostate, a gland in the male reproductive system below the bladder.
  • Risk factors for prostate cancer include age (especially after the age of 50; risk increases further after the age of 65, with -60% of prostate cancers found in men older than 65), ethnicity (prostate cancer is more common in African American men and in Caribbean men of African ancestry), family history (having a father or brother with prostate cancer more than doubles a man’s risk of developing the disease), and certain genetic variants or mutations (including variants of the BRCA1 and BRCA2 genes, and men with Lynch syndrome, a condition caused by inherited gene changes).
  • PSA prostate-specific antigen
  • prostate tumors remain small and cause no health problems. These are managed with active surveillance and monitoring tumor(s) with regular tests to ensure that have not grown. Tumors more likely to be dangerous can be targeted with radiation therapy or surgically removed by radical prostatectomy. Subjects whose cancer spreads beyond the prostate can be treated with hormone therapy, which reduces levels of the androgens (male sex hormones) that prostate cells need to survive. Cancer cells can eventually grow resistant to this treatment. This most-advanced stage of the disease, called castration-resistant prostate cancer (CRPC), can be treated with continued hormone therapy alongside with a chemotherapy drug (e g., docetaxel). Some tumors metastasize to other areas of the body, particularly the bones and lymph nodes.
  • a chemotherapy drug e g., docetaxel
  • Prostate cancer prognosis depends on how far the cancer has spread at diagnosis. Most men diagnosed have tumors confined to the prostate; 99% survive more than 10 years from their diagnoses. Tumors that have metastasized to distant body sites are most dangerous, with five- year survival rates of 30-40%.
  • PSA protein prostate-specific antigen
  • a typical man's blood has around 1 nanogram (ng) of PSA per milliliter (mb) of blood tested.
  • ng nanogram
  • mb milliliter
  • PSA levels below average are very unlikely to develop dangerous prostate cancer over the next 8 to 10 years.
  • Men with PSA levels above 4 ng/mL are at increased risk - around 1 in 4 will develop prostate cancer - and are often referred for a prostate biopsy. PSA levels over 10 ng/mL indicate an even higher risk: over half of men in this group develop prostate cancer.
  • Those with elevated PSA may undergo secondary screening blood tests that measure subtypes of PSA and other molecules to better predict the likelihood that a person will develop aggressive prostate cancer.
  • Many tests measure “free PSA” - the fraction of PSA unbound to other blood proteins, which is usually around 10% to 30%. Men who have a lower percentage of free PSA are more likely to have prostate cancer.
  • Several common tests more accurately detect prostate cancer cases by also measuring subtypes of free PSA, including the Prostate Health Index (measures a fragment called -2proPSA) and 4K score (measures intact free PSA).
  • Other tests measure blood levels of additional prostate-related proteins such as kallikrein-2 (also measured by 4K score), or urine levels of mRNA molecules common to prostate tumors like PC A3 and TMPRSS2 fused to ERG.
  • MRI magnetic resonance imaging
  • Prostate biopsies are typically taken by a needle passing through the rectum or perineum, guided by transrectal ultrasonography, MRI, or a combination of the two. Ten to twelve samples are taken from several regions of the prostate to improve the chances of finding any tumors. Biopsies are sent for a histopathologic diagnosis of prostate cancer, wherein they are examined under a microscope by a pathologist, who determines the type and extent of cancerous cells present. Cancers are first classified based on their appearance under a microscope.
  • adenocarcinomas (resembling gland tissue), with the rest largely squamous-cell carcinoma (resembling squamous cells, a type of epithelial cell) and transitional cell carcinoma (resembling transitional cells).
  • tumor samples are graded based on how much the tumor tissue differs from normal prostate tissue; the more different the tumor appears, the faster the tumor is likely to grow.
  • the Gleason grading system is commonly used, where the pathologist assigns numbers ranging from 3 (most similar to healthy prostate tissue) to 5 (least similar) to different regions of the biopsied tissue. They then calculate a “Gleason score” by adding the two numbers that represent the largest areas of the biopsy sample. The lowest possible Gleason score of 6 represents a biopsy most similar to healthy prostate; the highest Gleason score of 10 represents the most severely cancerous.
  • Gleason scores are commonly grouped into “Gleason grade groups”, which predict disease prognosis: a Gleason score of 6 is Gleason grade group 1 (best prognosis). A score of 7 (with Gleason scores 4 + 3, or Gleason scores 3 + 4, with the most prominent listed first) can be grade group 2 or 3; it is grade group 2 if the less severe Gleason score (3) covered more area; grade group 3 if the more severe Gleason score (4) covered more area. A score of 8 is grade group 4. A score of 9 or 10 is grade group 5 (worst prognosis).
  • the extent of cancer spread can be assessed by MRI or PSMA scan - a positron emission tomography (PET) imaging technique where a radioactive label that binds the prostate protein prostate-specific membrane antigen is used to detect metastases distant from the prostate.
  • PET positron emission tomography
  • CT scans may also be used but are less able to detect spread outside the prostate.
  • Bone scintigraphy can be used to test for spread of cancer to bones.
  • Prostate-specific membrane antigen is encoded by folate hydrolase 1 (FOLHI), and is a transmembrane glutamate carboxypeptidase that is highly expressed on prostate cancer cells. It consists of a large extracellular domain, a small transmembrane domain, and a cytoplasmic tail. High PSMA expression is a biomarker of poor prognosis throughout the course of prostate cancer and across anatomical sites. Metastatic lesions are PSMA-positive in most patients that have metastatic castration-resistant prostate cancer, and high PSMA expression has been independently associated with reduced survival.
  • FOLHI folate hydrolase 1
  • a PSMA PET scan is a nuclear medicine imaging technique that can be used in the diagnosis and staging of prostate cancer. It is carried out by injecting a radiopharmaceutical with a positron or gamma emitting radionuclide and a prostate-specific membrane antigen (PSMA) targeting ligand. After injection, imaging of positron emitters such as gallium-68 (68Ga), copper-64 (64Cu), and fluorine-18 (18F) is carried out with a positron emission tomography (PET) scanner. For gamma emitters such as technetium-99m (99mTc) and indium- 111 (11 Un) single-photon emission computed tomography (SPECT) imaging is performed with a gamma camera.
  • positron emitters such as gallium-68 (68Ga), copper-64 (64Cu), and fluorine-18 (18F) is carried out with a positron emission tomography (PET) scanner.
  • gamma emitters such as technetium-99
  • PSMA imaging can also be used to assess suitability for and plan treatment with external beam radiotherapy and PSMA- targeted therapeutics (e.g., PSMA-targeted radionuclides).
  • PSMA- targeted therapeutics e.g., PSMA-targeted radionuclides.
  • PSMA targeting therapies such as radionuclide therapies (e.g., lutetium- 177 (177Lu)-PSMA-617) can target prostate cancer cells while sparing most normal tissues in patients who have been selected with the use of imaging to confirm radionuclide binding.
  • radionuclide therapies e.g., lutetium- 177 (177Lu)-PSMA-617
  • PSMA expression in tumors is commonly assessed using PSMA- targeted positron emission tomography (PET), which has gained increased acceptance in diagnosing prostate cancer due to its superior accuracy in identifying metastases as compared to CT and MRI methods.
  • PET positron emission tomography
  • Patient eligibility for lutetium- 177 (177Lu)-PSMA-617 currently requires PSMA PET imaging.
  • MEDI3726 an engineered version of an anti-PSMA IgGlK antibody (J591), site- specifically conjugated with pyrrolobenzodiazepine (PBD) dimers (SG3199). Described, e.g., in de Bono et al. “Phase I study of MEDI3726: a prostate-specific membrane antigen-targeted antibody-drug conjugate, in patients with mCRPC after failure of abiraterone or enzalutamide.” Clinical Cancer Research 27.13 (2021): 3602-3609.
  • PSA Prostate-specific antigen
  • KLK3 kallikrein- 3
  • P-30 antigen is a glycoprotein enzyme encoded in humans by the KLK3 gene.
  • PSA is a member of the kallikrein-related peptidase family and is secreted by the epithelial cells of the prostate gland in men and the paraurethral glands in women.
  • PSA levels can also be monitored (e.g., measured periodically (e.g., every 6-36 months)).
  • patients with high-risk disease are monitored at an increased frequency as compared to patients with lower-risk disease.
  • surgical therapy i.e., radical prostatectomy
  • PSA can become undetectable within a few weeks.
  • a subsequent rise in PSA level above 0.2 ng/mL is generally regarded as evidence of recurrent prostate cancer after a radical prostatectomy; less commonly, it may simply indicate residual benign prostate tissue.
  • Recurrent prostate cancer detected by a rise in PSA levels after curative treatment is referred to as a “biochemical reoccurrence.”
  • the likelihood of developing recurrent prostate cancer after curative treatment is related to the pre-operative variables described in the preceding section (PSA level and grade/stage of cancer).
  • Low-risk cancers are the least likely to recur, but they are also the least likely to have required treatment in the first place.
  • a sample analyzed using methods, kits and systems provided herein can be any biological sample including any processed sample that includes cell free DNA (cfDNA) derived from a biological sample.
  • a sample analyzed using methods, kits and systems provided herein can be a sample obtained from a mammalian subject.
  • a sample analyzed using methods, kits and systems provided herein can be a sample obtained from a human subject.
  • a sample from a subject can be obtained from a liquid biopsy.
  • a sample and/or reference is obtained from serum, plasma, or urine.
  • the sample is serum.
  • a sample comprises cell free DNA (cfDNA).
  • cfDNA cell free DNA
  • a sample is derived from about 1 mL of blood obtained from the subject.
  • a sample is derived from about 0.5-2 mL of blood obtained from the subject, e.g., about 0.5 to 1 .75 mL, about 0.5 to 1.5 mb, about 0.75 to 1.25 mL or about 0.9 to 1.1 mL of blood.
  • a sample comprises circulating tumor DNA (ctDNA).
  • a sample is derived from about 1 mL of blood obtained from the subject.
  • a sample is a sample of cell-free DNA (cfDNA).
  • cfDNA is typically found in human biofluids (e.g., plasma, serum, or urine) in short, double-stranded fragments.
  • the concentration of cfDNA is typically low, but can significantly increase under particular conditions, including without limitation pregnancy, autoimmune disorders, myocardial infarction, and cancer.
  • Circulating tumor DNA is a component of cell-free DNA specifically derived from cancer cells.
  • ctDNA can be present in human biofluids bound to leukocytes and erythrocytes or not bound to leukocytes and erythrocytes.
  • ctDNA comprises less than 30%, less than 20%, or less than 10% of the cfDNA in the liquid biopsy sample obtained from the subject, e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or less than 1% of the cfDNA in the sample.
  • the percentage of ctDNA in the liquid biopsy sample is assessed using ichorCNA which estimates the percentage of ctDNA in a sample probabilistically (see Adalsteinsson et al., Nat Commun (2017) 8(1): 1324 the entire contents of which are incorporated herein by reference).
  • cfDNA and ctDNA can provide a real-time or nearly real time metric of status of a source tissue.
  • cfDNA and ctDNA demonstrate a half-life in blood of about 2 hours, such that a sample taken at a given time provides a relatively timely reflection of the status of a source tissue.
  • Samples include materials prepared by processes including, without limitation, steps such as concentration, dilution, adjustment of pH, removal of high abundance polypeptides (e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives, addition of calibrants, addition of protease inhibitors, addition of denaturants, desalting, concentration and/or extraction of sample nucleic acids, and/or amplification of sample nucleic acids (e.g., by PCR or other nucleic acid amplification techniques). Samples also include materials prepared by techniques that isolate, e.g., nucleosomes or transcription factors and/or nucleic acids associated with nucleosomes or transcription factors.
  • steps such as concentration, dilution, adjustment of pH, removal of high abundance polypeptides (e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives, addition of calibrants, addition of protease inhibitors, addition of denaturants
  • Removal from a sample of proteins that are not desirable for a relevant purpose or context can be achieved using high affinity reagents, high molecular weight filters, ultracentrifugation and/or electrodialysis.
  • High affinity reagents include antibodies or other reagents (e.g., aptamers) that selectively bind to high abundance proteins.
  • Sample preparation can also include ion exchange chromatography, metal ion affinity chromatography, gel filtration, hydrophobic chromatography, chromatofocusing, adsorption chromatography, isoelectric focusing and related techniques.
  • the membranes used in electrodialysis may have the ability to selectively transport ions having positive or negative charge, reject ions of the opposite charge, or to allow species to migrate through a semipermeable membrane based on size and charge, it renders electrodialysis useful for concentration, removal, or separation of electrolytes.
  • Separation and purification in the present disclosure may include any procedure known in the art, such as capillary electrophoresis (e.g., in capillary or on-chip) or chromatography (e.g., in capillary, column or on a chip).
  • Electrophoresis is a method that can be used to separate ionic molecules under the influence of an electric field. Electrophoresis can be conducted in a gel, capillary, or in a microchannel on a chip. Examples of gels used for electrophoresis include starch, acrylamide, polyethylene oxides, agarose, or combinations thereof.
  • a gel can be modified by its cross-linking, addition of detergents, or denaturants, immobilization of enzymes or antibodies (affinity electrophoresis) or substrates (zymography) and incorporation of a pH gradient.
  • capillaries used for electrophoresis include capillaries that interface with an electrospray.
  • CE Capillary electrophoresis
  • CZE capillary zone electrophoresis
  • CIEF capillary isoelectric focusing
  • CITP capillary isotachophoresis
  • CEC capillary electrochromatography
  • An embodiment to couple CE techniques to electrospray ionization involves the use of volatile solutions, for example, aqueous mixtures containing a volatile acid and/or base and an organic such as an alcohol or acetonitrile.
  • Capillary isotachophoresis is a technique in which the analytes move through the capillary at a constant speed but are nevertheless separated by their respective mobilities.
  • Capillary zone electrophoresis also known as free-solution CE (FSCE)
  • FSCE free-solution CE
  • Capillary isoelectric focusing allows weakly-ionizable amphoteric molecules, to be separated by electrophoresis in a pH gradient.
  • CEC is a hybrid technique between traditional high performance liquid chromatography (HPLC) and CE.
  • Separation and purification techniques used in the present disclosure can include any chromatography procedures known in the art. Chromatography can be based on the differential adsorption and elution of certain analytes or partitioning of analytes between mobile and stationary phases. Different examples of chromatography include, but not limited to, liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (HPLC), etc.
  • LC liquid chromatography
  • GC gas chromatography
  • HPLC high performance liquid chromatography
  • whole blood is collected from a subject, and a plasma layer is separated by centrifugation.
  • cfDNA may be then extracted from the plasma using methods known in the art.
  • Histone methylation is understood to increase or decrease expression of associated coding sequences, depending on which histone residue is methylated.
  • Histone methylation is an essential modification that can cause monomethylation (mel), dimethylation (me2), and trimethylation (me3) of several amino acids, thus directly affecting heterochromatin formation, gene imprinting, X chromosome inactivation, and gene transcriptional regulation.
  • Histone methyltransferases promote monomethylation, dimethylation, or trimethylation of histones while histone demethylases promote demethylation of histones.
  • Histone methylation In general, lysine (Lys or K), arginine (Arg or R), and rarely histidine (His or H) are the most common histone methyl acceptors. Histone methylation only occurs at specific lysine and arginine sites of histone H3 and H4. In histone H3, lysine 4, 9, 26, 27, 36, 56, and 79 and arginine 2, 8, and 17 can be methylated. By comparison, histone H4 has fewer methylation sites, in which only lysine 5, 12, and 20 and arginine 3 can be methylated. Histone methylation is often associated with transcriptional activation or inhibition of downstream genes.
  • H3K4, R8, R17, K26, K36, K79, H4R3, and K12 can activate gene transcription.
  • the methylation of histone H3K9, K27, K56, H4K5, and K20 can inhibit gene transcription.
  • H3K4 methylation generally activates gene expression
  • H3K27 methylation generally represses gene expression.
  • Histone acetylation occurs predominantly at lysine residues and is generally understood to increase expression of associated coding sequences. Without wishing to be bound by any theory, acetylation of lysine residues is thought to neutralize lysine’s positive charge and thereby cause histones to drift away from DNA, which has a negative charge. The released structure facilitates access to transcriptional machinery such as transcription factors and RNA polymerase II. Histone acetylation and deacetylation are generally catalyzed by histone acetyltransferases (HATs) and HDACs, respectively. Acetyl-CoA can be a source and co-factor of acetylation.
  • HATs histone acetyltransferases
  • HDACs histone acetyltransferases
  • HATs can acetylate histones and recruit HAT-containing complexes to activate the transcriptional process.
  • H3K9ac and H3K27ac levels can be associated with promoter and enhancer activities.
  • H3K27ac enhances not only the kinetics of transcriptional activation, but also accelerates the transition of RNA polymerase II from the initiation state to the elongation state.
  • Differential modification of a genomic locus can refer to, or be determined by or detected as, a comparative difference or change in modification status of one or more genomic loci between a first sample, condition, disease, or state and a second or reference sample, condition, disease, or state.
  • a reference is typically produced by measurement using a methodology identical, similar, or comparable to that by which a compared non-reference measurement was taken.
  • Chromatin accessibility can refer to the degree to which nuclear macromolecules are able to physically contact DNA and is determined in part by the occupancy and modification status of nucleosomes.
  • Modified histones can regulate chromatin accessibility through a variety of mechanisms, such as altering transcription factor (TF) binding through steric hindrance and modulating nucleosome affinity for active chromatin remodelers.
  • TF transcription factor
  • the topological organization of nucleosomes across the genome is non-uniform: while histones can be densely arranged within facultative and constitutive heterochromatin, histones can be depleted at regulatory loci, including within enhancers, insulators and transcribed gene bodies. Active regulatory elements of the genome are generally accessible.
  • Differential accessibility of a genomic locus can refer to, or be determined by or detected as, a comparative difference or change in modification status of one or more genomic loci between a first sample, condition, disease, or state and a second or reference sample, condition, disease, or state.
  • a reference is typically produced by measurement using a methodology identical, similar, or comparable to that by which a compared non-reference measurement was taken.
  • a reference can be a value or set of values that are predetermined or derived from a sample or set of samples.
  • a reference can be a sample or set of samples.
  • a reference value can be a predetermined threshold value, a value that varies in accordance with circumstances (e.g., according to patient subpopulation, age, weight, or other variables), or a ratio.
  • Reference ratios can be ratios relating to the modification and/or accessibility of multiple loci within individual samples and/or references, or across or between samples and/or references.
  • a reference can have or represent a normal, non-diseased state.
  • a reference can have or represent a diseased state, e.g., prostate cancer.
  • a reference can represent prostate cancer by being obtained from a subject diagnosed as having prostate cancer (e.g., based on imaging, symptoms, and/or biomarker analysis).
  • a reference is a non-contemporaneous sample from the same source, e.g., a prior sample from the same source, e.g., from the same subject.
  • a reference for the modification status of one or more genomic loci can be the modification status of the one or more genomic loci (e.g., one or more differentially modified genomic loci) in a sample (e.g., a sample from a subject), or a plurality of samples, known to represent a particular state (e.g., mCRPC with elevated PSMA expression or prostate cancer with elevated serum PSA).
  • a reference for the accessibility status of one or more genomic loci can be the accessibility status of the one or more genomic loci (e.g., one or more differentially accessible genomic loci) in a sample (e.g., a sample from a subject), or a plurality of samples, known to represent a particular state (e.g., mCRPC with elevated PSMA expression or prostate cancer with elevated serum PSA).
  • differential modification or differential accessibility can refer to a differential (e.g., between a sample and a reference) with an absolute log2(fold-change) that is greater than or equal to 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 or more, or any range in between, inclusive, e.g., as measured according to an assay provided herein.
  • Enhancers are genomic loci that can be differentially modified or differentially accessible in and/or between conditions, diseases, and other states. Enhancers are cis-acting DNA regulatory regions that are thought to bind trans-acting proteins that contribute to expression patterns of associated genes. Chromatin ImmunoPrecipitation sequencing (ChlP-seq) of histone modifications (e.g., acetylation) have identified millions of enhancers in mammalian genomes. The number of active enhancers in any given cell type is estimated to be in the tens of thousands. Certain transcription factors (TFs), sometimes referred to as “master” transcription factors, associate with active enhancers with important impacts on gene expression and cell function.
  • TFs transcription factors
  • transcription factors preferentially associate with enhancers that regulate genes required for establishing cell identity and function, including enhancer domains known as “super-enhancers”.
  • master TFs can participate in inter-connected auto-regulatory circuitries or “cliques” that are self-reinforcing, show marked cell selectivity, and function to maintain cell state and/or cell survival.
  • Chromatin ImmunoPrecipitation is one technique of molecular biology useful in detecting and quantifying histone modifications and transcription factor binding in samples.
  • CUT&RUN or CUT&Tag are other more recent techniques that can also be used to detect and quantify histone modifications and transcription factor binding sites.
  • ChIP -chip, ChlP-exo, ChIP Re-ChIP, and ChlPmentation are other alternative techniques that could be used.
  • ChIP can involve various steps including one or more of fixation, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA.
  • ChIP has become a very widely used tissue-based technique for determining the in vivo location of binding sites of various transcription factors and histones. Because the proteins are captured at the sites of their binding with DNA, ChIP helps to detect DNA-protein interactions that take place in living cells. More importantly, ChIP can be coupled to many commonly used molecular biology techniques such as PCR and real-time PCR, PCR with single-stranded conformational polymorphism, Southern blot analysis, Western blot analysis, cloning, and microarray. The resulting versatility has increased the potential of this technique.
  • ChIP of tissue samples usually involves cross-linking of the chromatin-bound proteins by formaldehyde, followed by sonication or nuclease treatment to obtain small DNA fragments. Immunoprecipitation can be then carried out using specific antibodies to the DNA- binding protein of interest. The DNA can be then released from the proteins and analyzed using various methods. ChIP has also been used to study RNA-protein interactions. X-ChIP methods utilize fixed chromatin fragmented by sonication, while the N-ChIP methods utilize native chromatin, which can be unfixed and nuclease digested.
  • the first step of the technique can be the cross-linking of DNA and proteins.
  • Formaldehyde is one of the most used cross-linking agents.
  • One advantage of using formaldehyde can be the ease of reversibility of the cross-links and its ability to form bonds that span approximately 2 angstroms. This means that formaldehyde can bind molecules in close association with each other.
  • formaldehyde can be added to the medium in the cell culture flask or plate. It enters the cells through the cell membrane and cross-links the proteins to the chromatin. Formaldehyde fixation of tumor tissues has also been done.
  • Other cross-linking agents that have been used include chemicals such as methylene blue and acridine orange, cisplatin, dimethylarsinic acid, potassium chromate, and ultraviolet (UV) light and lasers.
  • Harvested chromatin can be sonicated in one or more sonication cycles.
  • DNA can be typically broken into to 100-500 bp fragments to pinpoint the location of the DNA sequence of interest.
  • An alternative to sonication can be nuclease digestion of the chromatin, e.g., in N- ChlP methods.
  • Purification of chromatin can be achieved using a cesium chloride (CsCl) gradient centrifugation.
  • CsCl cesium chloride
  • Chromatin can be immunoprecipitated using one or more antibodies that bind a target epitope.
  • an antibody used in ChIP can selectively bind a particular transcription factor or one or more particular histone modifications, such as one or more particular histone acetylation modifications or histone methylation modifications.
  • an antibody used to bind a target epitope can be a “pan” antibody (e.g., a panacetylation antibody, a pan-methylation antibody, an antibody that binds a group of histone modifications associated with increased transcription activation, and/or an antibody that binds a group of histone modifications associated with increased transcription repression).
  • the antibody against the protein of interest is allowed to bind to the protein-DNA complex, and the complex can be then precipitated.
  • Immunosorbants commonly used to separate the antigen-antibody complex from the lysate include salmon sperm DNA-protein A-Sepharose®, protein G, magnetic beads, and other engineered immunoprecipitation systems known to those of skill in the art.
  • Immunoprecipitated DNA can be eluted. Once the DNA of interest is isolated, many detection and quantification methods can be used to study the isolated gene fragments. Commonly utilized methods include PCR, real-time PCR, slot blot hybridization, microarray techniques, and deep or next-generation sequencing. ChlP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. ChlP-seq can be used to map DNA-binding proteins, e.g., transcription factor binding sites and histone modifications in a genome-wide manner.
  • ChIP chromatin immunoprecipitation
  • Cell-free Chromatin ImmunoPrecipitation sequencing involves applying ChlP-seq to samples that include cell-free DNA, e.g., liquid biopsy samples including cfDNA such as plasma samples including cfDNA (e.g., see Sadeh et al., Nat Biotechnol (2021) 39: 586-598 and Jang et al., Life Sci Alliance (2023) 6(12):e202302003 the entire contents of each of which are incorporated herein by reference).
  • cfChlP-seq uses antibodies or antibody fragments that bind specific histone modifications (e.g., H3K4me3 and/or H3K27ac) and/or transcription factors that are coupled (covalently or non-covalently) to beads, e g., magnetic beads such as Dynabeads® magnetic beads and incubated with a volume, e.g., about 1 mL of thawed plasma obtained from a subject.
  • specific histone modifications e.g., H3K4me3 and/or H3K27ac
  • transcription factors that are coupled (covalently or non-covalently) to beads, e g., magnetic beads such as Dynabeads® magnetic beads and incubated with a volume, e.g., about 1 mL of thawed plasma obtained from a subject.
  • exemplary antibodies that bind H3K4me3 include PA5-27029 (available from Thermo Fisher Scientific in Waltham, MA) and C15410003 (available from Diagenode in Denville, NJ) and exemplary antibodies that bind H3K27ac include ab21623 or ab4729 (both available from Abeam in Cambridge, UK) and Cl 5210016 (available from Diagenode in Denville, NJ).
  • the antibodies or antibody fragments can be covalently coupled to beads, e.g., epoxy beads.
  • the antibodies or antibody fragments can be non-covalently coupled to beads, e.g., Protein A or Protein G beads such as Dynabeads® Protein A or Dynabeads® Protein G beads.
  • a cfDNA library is then typically prepared from the captured cfDNA. Library preparation can be done on-bead or after releasing the captured cfDNA by digestion of bound histones, e g., using proteinase K.
  • the cfDNA library is then sequenced to generate reads of captured cfDNA sequences, e.g., by next-generation sequencing (NGS) as is known in the art.
  • NGS next-generation sequencing
  • the reads are then analyzed, e.g., aligned and counted using standard bioinformatic techniques as is known in the art.
  • a cfChlP-seq bioinformatic pipeline can include, e.g., alignment of sequence reads to a reference genome with BWA or Bowtie2. Aligned reads can be used to call and quantify peaks as compared to a reference.
  • CUT&Tag involves antibody-based binding of a target protein, e.g., transcription factor or histone modification of interest, where antibody incubation is directly followed by the shearing of the chromatin and library preparation (see Kaya-Okur et al., Nat Comm (2019) 10: 1930).
  • CUT&Tag assays take advantage of a Tn5 transposase that is fused with Protein A to direct the enzyme to the antibody bound to its target on chromatin.
  • Tn5 transposase is pre-loaded with sequencing adapters (generating the assembled pA-Tn5 adapter transposome) to carry out antibody-targeted tagmentation.
  • samples are incubated with an antibody immobilized on Concanavalin A-coated magnetic beads to facilitate subsequent washing steps.
  • Cells can be incubated with a primary antibody specific for the target protein of interest followed by incubation with a secondary antibody.
  • Samples can then be incubated with assembled transposomes, which consist of Protein A fused to the Tn5 transposase enzyme that is conjugated to NGS adapters. After incubation, unbound transposome can be washed away using stringent conditions.
  • Tn5 is a Mg 2+ -dependent enzyme so Mg 2+ can be added to activate the reaction, which results in the chromatin being cut close to the protein binding site and simultaneous addition of the NGS adapter DNA sequences. Chromatin cleavage and library preparation can be achieved in one single step.
  • CUT&RUN is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing (see Skene and Henikoff, Elife (2017) 6:1-35, Skene et al., Nat Protoc (2016) 13:1006-1019). As only targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has low background levels.
  • a sample is incubated with an antibody or antibody fragment that binds the target protein, e.g., transcription factor or histone modification of interest.
  • the sample is then incubated with Protein-A-MNase after which CaCh can be added to initiate the calcium dependent nuclease activity of MNase to cleave the DNA around the target protein.
  • the protein- A-MNase reaction can be quenched by adding chelating agents (EDTA and EGTA). Cleaved DNA fragments are then liberated, extracted, and used to construct a sequencing library.
  • kits and systems of the present disclosure involve the detection and quantification of chromatin accessibility in samples, e.g., in liquid biopsy samples including cfDNA such as plasma samples including cfDNA.
  • ATAC-seq Assay of Transpose Accessible Chromatin sequencing
  • NOMe-seq Nucleosome Occupancy and Methylome sequencing
  • FAIRE-seq Formmaldehyde-Assisted Isolation of Regulatory Elements sequencing
  • MNase-seq Merococcal Nuclease digestion with sequencing
  • DNase hypersensitivity assays are exemplary techniques of molecular biology useful in detecting and quantifying chromatin accessibility in samples.
  • Sono-Seq is another alternative method that could be used (see Auerbach et al., Proc Natl Acad USA (2009) 106(35): 14926-14931).
  • Fragmentomics-based methods are yet another method that can be used to assess chromatin accessibility (see Ding, Spencer C., and YM Dennis Lo. "Cell-free DNA fragmentomics in liquid biopsy.” Diagnostics 12.4 (2022): 978).
  • DNase hypersensitivity assays can use the non-specific DNA endonuclease Deoxyribonuclease I (DNase I), which selectively digests accessible DNA regions.
  • DNase I hypersensitivity sites (DHS) identified by DNase-seq include open chromatin regulatory regions.
  • a typical DNase hypersensitivity assay can include a first step in which nuclei are isolated from cells using lysis buffer, and nuclei are digested using DNase I. DNA fragment sizes are measured to identify optimal digestion using gel electrophoresis. Biotinylated linkers can be ligated to the ends of digested DNA after polishing to make blunt ends, and the DNA can then be isolated.
  • DNA with biotinylated linker can be digested by restriction endonuclease Mmel and captured by streptavidin coated Dynabeads® to generate short tags to which a second sequencing adaptor can be ligated.
  • a second linker can be ligated and amplified to generate a library for sequencing.
  • a DNase-seq bioinformatic pipeline can include, e.g., alignment of sequence reads to a reference genome with BWA or Bowtie2. Aligned reads can be used to call and quantify peaks as compared to a reference.
  • MNase-seq determines chromatin accessibility with micrococcal nuclease (MNase) that preferentially digests nucleosome-free, protein-unbound DNA.
  • MNase- seq assay can include a first step in which nuclei are isolated from either native or crosslinked chromatin and digested using MNase with titration. In vivo formaldehyde crosslinking step that is designed to capture the interaction between proteins and DNA. This crosslinking allows bound proteins to shield their associated DNA from digestion by MNase. Following crosslinking, samples are digested with MNase, which can be specifically activated by addition of Ca2+ to the buffer.
  • Digestion can be halted by chelating the reaction, at which point the samples are RNase treated, crosslinks are reversed, and proteins are digested away from the chromatin. DNA can then be isolated via a phenol-chloroform extraction. Uncut DNA is purified and mononucleosome bands are isolated and excised through gel electrophoresis. Isolated DNA can be amplified by adding adapters to generate a library, and sequenced. MNase-seq primarily sequences regions of DNA bound by histones or other proteins. Therefore, it indirectly determines which regions of DNA are accessible by directly determining which regions are bound to nucleosomes or proteins.
  • FAIRE-seq is a method in which nucleosome-depleted regions of DNA (NDRs) are isolated from chromatin.
  • a typical FAIRE-seq assay can include a first step in which cells are fixed using formaldehyde so that histones are crosslinked to interacting DNA. Crosslinked chromatin can then be sheared by sonication that generates protein-free DNA and protein- crosslinked DNA fragments. Protein-free DNA can be isolated using a phenol-chloroform extraction: DNA crosslinked with protein stays in organic phase, while protein-free DNA stays in aqueous phase. Highly crosslinked DNA remains in the organic phase and the non-crosslinked DNA is pulled to the aqueous phase.
  • Non-crosslinked DNA from the aqueous phase can then be amplified and sequenced. Reads enriched in the sequencing pool tend to have lower nucleosome and transcription factor binding and are therefore inferred to come from accessible regions.
  • NOMe-seq is a method to identify nucleosome-depleted regions of DNA (NDRs) with M.CviPI methyltransferase that methylates cytosine in GpC dinucleotides not protected by nucleosomes or other proteins.
  • NDRs nucleosome-depleted regions of DNA
  • M.CviPI methyltransferase M.CviPI methyltransferase that methylates cytosine in GpC dinucleotides not protected by nucleosomes or other proteins.
  • GpC m in the human genome does not occur naturally in most cell types. GpC in levels at open chromatin regions can be compared to background signals and used to detect and quantify NDRs.
  • a typical NOMe-seq protocol can include a step in which samples are treated with M.CviPI and S-adenosylhomocysteine (SAM) to methylate accessible GpC sites.
  • M.CviPI treated DNA can be sheared using a sonicator, so that DNA fragments can be sequenced.
  • DNA is treated with bisulfite, which converts unmethylated cytosine to uracil using sodium bisulfite, while methylated cytosine is unaffected.
  • a library is generated using adapters and sequenced. Accessible chromatin is expected to have high levels of GpC m but low levels of C m pG. Therefore, NOMe-seq identifies NDRs using the two separate methylation analyses that serve as independent (but opposite) measures, providing matched chromatin designations for each regulatory element.
  • RRBS Reduced representation bisulfite sequencing
  • DNA methylation typically refers to the methylation of the 5’ position of cytosine (mC) by DNA methyltransferases (DNMT). It is a major epigenetic modification in humans and many other species. In mammals, most DNA methylations occur within the context of CpG dinucleotides. DNA methylation is thought to be a repressive chromatin modification. Aberrant methylation can lead to many diseases including cancers (Robertson, Nat Rev Genet (2005) 6:597-610 and Bergman and Cedar, Nat Struct Mol Biol (2013) 20:274-281).
  • MeDIP-seq was first reported by Weber et al., Nat Genet (2005) 37:853-862.
  • antibody or antibody-fragment that binds 5-methylcytidine (5mC) is used to enrich methylated DNA fragments, then these fragments are sequenced and analyzed. If using 5mC-specific antibodies or antibody fragments, methylated DNA is isolated from genomic DNA via immunoprecipitation. Anti-5mC antibodies are incubated with fragmented genomic DNA and precipitated, followed by DNA purification and sequencing.
  • Methyl-CpG-Binding Domain sequencing is similar to MeDIP-seq except that it uses methyl binding domain (MBD) proteins instead of antibodies or antibody fragments to bind methylated DNA.
  • MBD methyl binding domain
  • genomic DNA is first sonicated and incubated with tagged MBD proteins that can bind methylated cytosines.
  • the protein-DNA complex is then precipitated with antibody -conjugated beads that are specific to the MBD protein tag, followed by DNA purification and sequencing.
  • a subject is determined to have an epigenetic profile indicative of a cancer associated with elevated serum PSA levels based on analysis of a biological sample, optionally of cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject.
  • cfDNA cell-free DNA
  • a cancer is determined to be PSMA-positive if PSMA expression is detected that is above a threshold value.
  • the threshold value is a predetermined threshold and/or a normalized value.
  • the threshold value is a PSMA expression level determined in a reference population.
  • the reference population comprises subjects having prostate cancer and previously found to respond to treatment with a PSMA-targeted therapeutic.
  • the reference population comprises subjects having cancer and previously found to not respond to treatment with a PSMA-targeted therapy.
  • the reference population comprises subjects having a PSMA-positive cancer (e.g., as determined by PSMA PET imaging).
  • the reference population comprises subjects having a low PMSA expressing cancer (e.g., as determined by PSMA PET imaging) or subjects with a cancer having a level of PSMA expression that is associated with poor response to a PSMA-targeted therapeutic. In some embodiments, the reference population comprises subjects determined to be cancer free.
  • PSA expression is determined or predicted to be elevated if the determined or predicted value is above a threshold value.
  • the threshold value is a predetermined threshold and/or a normalized value.
  • the threshold value is a PSA expression level determined in a reference population.
  • the reference population comprises subjects having prostate cancer.
  • the reference population comprises subjects that have not been diagnosed with cancer.
  • the present disclosure is not limited to methods that use the exact same chromosomal coordinates that are recited in Tables 1-5.
  • the present disclosure encompasses methods that use any of the genomic loci in Tables 1-5 and also subregions thereof, i.e., references herein to methods that involve detecting and/or quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci of Tables 1-5 encompasses methods that detect these marks anywhere within these genomic loci including within any subregions.
  • a classifier is generated using a set of differentially modified and/or differentially accessible genomic loci that are correlated with increased PSA or PSMA expression (e.g., increased PSMA PET SUVmean or increased PSA concentrations). Sequence reads that fall into each selected genomic locus are analyzed and counted, e.g., as described herein including the Examples. In some embodiments, counts from genomic loci that are correlated with increased PSMA or PSA expression (e.g., increased PSMA PET SUVmean or increased serum PSA) are aggregated.
  • exemplary genomic loci from one or more of Tables 1-5 are used in a monomodal PSMA PET Score Model Predictor, e.g., a PSMA PET Score Model Predictor that uses a single histone modification (e.g., H3K4me3 or H3K27ac) or DNA methylation at one or more genomic loci for purposes of determining PSMA expression level.
  • a monomodal PSMA PET Score Model Predictor e.g., a PSMA PET Score Model Predictor that uses a single histone modification (e.g., H3K4me3 or H3K27ac) or DNA methylation at one or more genomic loci for purposes of determining PSMA expression level.
  • exemplary genomic loci from any one of Table 1-5, or any combination thereof are used in combination in a multimodal classifier, e.g., a PSMA PET Score Model Predictor that uses more than one histone modification (e.g., H3K4me3 and H3K27ac) or one or more histone modifications (e.g., H3K4me3 and/or H3K27ac) and DNA methylation at one or more genomic loci for purposes of measuring PSMA expression.
  • a multimodal classifier e.g., a PSMA PET Score Model Predictor that uses more than one histone modification (e.g., H3K4me3 and H3K27ac) or one or more histone modifications (e.g., H3K4me3 and/or H3K27ac) and DNA methylation at one or more genomic loci for purposes of measuring PSMA expression.
  • a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and/or binding of a transcription factor at one or more loci provided in one or more of Tables 1-5. In some embodiments, a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and/or binding of a transcription factor at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 loci listed in one or more of Tables 1-5.
  • a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and/or binding of a transcription factor at each of the loci provided in Table 1, Table 2, Table 3, Table 4, and/or Table 5. In some embodiments, a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and/or binding of a transcription factor for at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Tables 1-5, or any combination thereof.
  • a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and/or binding of a transcription factor for at least a percent of loci identified in Table 1, Table 2, Table 3, Table 4, and/or Table 5 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100%.
  • Exemplary genomic loci whose H3K4 methylation state (in particular H3K4 trimethylation, H3K4me3) is associated with PSMA expression level are provided in Tables 1, 2, 4 and 5 (see H3K4me3 analyte loci).
  • Exemplary genomic loci whose H3K4 methylation state (in particular H3K4 trimethylation, H3K4me3) is associated with PSA expression level are provided in Tables 3.
  • Subsets of the H3K4me3 analyte genomic loci of Tables 1-5 can be selected (e.g., for use in determining PSMA expression level or PSA expression level) based on various performance criteria, e.g., to select genomic loci that demonstrate differential modification with a particular level of statistical significance and/or a particular threshold of differential between relevant states (e.g., a measured log2(fold-change)). Subsets of the genomic loci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier.
  • a sample or subject from which the sample is obtained or derived is determined to have a particular PSMA expression level if one or both H3K4me3 analyte loci identified in Table 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from (i) a healthy subject or cohort of healthy subjects or (ii) a subject with aberrant PSMA expression or a cohort of subjects with aberrant PSMA expression (e.g., a subject or cohort of subjects with mCRPC and that exhibit a positive PSMO PET SUVmean signal)).
  • a reference e.g., a sample from (i) a healthy subject or cohort of healthy subjects or (ii) a subject with aberrant PSMA expression or a cohort of subjects with aberrant PSMA expression (e.g., a subject or cohort of subjects with mCRPC and that exhibit a positive PSMO PET SUVmean signal)).
  • a sample or subject from which the sample is obtained or derived is determined to have a particular PSMA expression level if the H3K4me3 analyte loci identified in Table 5 is differentially H3K4me3 modified as compared to a reference (e.g., a sample from (i) a healthy subject or cohort of healthy subjects or (ii) a subject with aberrant PSMA expression or a cohort of subjects with aberrant PSMA expression (e.g., a subject or cohort of subjects with mCRPC and that exhibit a positive PSMO PET SUV mean signal)).
  • a reference e.g., a sample from (i) a healthy subject or cohort of healthy subjects or (ii) a subject with aberrant PSMA expression or a cohort of subjects with aberrant PSMA expression (e.g., a subject or cohort of subjects with mCRPC and that exhibit a positive PSMO PET SUV mean signal).
  • a sample or subject from which the sample is derived is determined to have a particular PSMA expression level if one or more promoter regions of one or more H3K4me3 analyte genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) in Table 1 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from (i) a healthy subject or cohort of healthy subjects or (ii) a subject with aberrant PSMA expression or a cohort of subjects with aberrant PSMA expression (e.g., a subject or cohort of subjects with mCRPC and that exhibit a positive PSMO PET SUVmean signal)).
  • a reference e.g., a sample from (i) a healthy subject or cohort of healthy subjects or (ii) a subject with aberrant PSMA expression or a cohort of subjects with aberrant PSMA expression (e.g., a subject or cohort of subjects with mCRPC and that exhibit a positive PSMO PET SUVmean signal)).
  • a promoter region refers to a region a certain number of nucleotides upstream of a gene (e.g., 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, or 1,000 nucleotides upstream of a gene). In some embodiments, a promoter region refers to a region identified in any one of Tables 1-5.
  • a sample or subject from which the sample is obtained or derived is determined to have a particular PSMA expression level if H3K4me3 modifications for 1, 2, 3, 4, 5, 6, or 7 of the H3K4me3 analyte loci that are identified in Table 1 as having a positive association with PSMA expression are increased relative to a reference (e.g., a sample from (i) a healthy subject or cohort of healthy subjects or (ii) a subject with aberrant PSMA expression or a cohort of subjects with aberrant PSMA expression (e.g., a subject or cohort of subjects with mCRPC and that exhibit a positive PSMO PET SUVmean signal)).
  • a reference e.g., a sample from (i) a healthy subject or cohort of healthy subjects or (ii) a subject with aberrant PSMA expression or a cohort of subjects with aberrant PSMA expression (e.g., a subject or cohort of subjects with mCRPC and that exhibit a positive PSMO PET SUVmean signal)).
  • differentially H3K4me3 modified refers to a methylation status characterized by an increase or decrease in a value measuring methylation (e.g., of read counts and/or normalized read counts for a given genomic locus), and/or a mean, median and/or mode thereof, and/or a log thereof e.g., log base 2 (log2)), of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40- fold, 45-fold, 50-fold, or greater, or any range in between, inclusive, such as 1% to 50%, 50% to 2-fold, 25% to 50-fold, 25% to 30-fold, 25% to 20-fold
  • an increase or decrease in a value measuring methylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or greater, or any range in between, inclusive, such as an increase or decrease of 0.1 -fold to 10- fold, 0.2-fold to 5-fold, 0.2-fold to 4.0-fold, 0.4-4.0-fold, 0.4-fold to 4.0-fold, 0.6-fold to 4.0- fold, 0.8-fold to 4.0-fold, 1.0-fold to 4.0-fold.
  • a log2(fold-change) e.g., a log2(fold-change
  • H3K27ac analyte genomic locus listed in Tables 1-5 be assessed for H3K27ac modifications. Instead, a subset of H3K27ac analyte loci may be assessed for H3K27ac modification. Subsets of the H3K27ac analyte genomic loci of Tables 1-5 can be selected (e.g., for use in determining PSMA expression level) based on various performance criteria, e.g., to select genomic loci that demonstrate differential modification with a particular level of statistical significance and/or a particular threshold of differential between relevant states (e.g., a measured log2(fold-change)).
  • Subsets of the genomic loci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier.
  • an algorithm e.g., during the process of obtaining a classifier.
  • loci of Tables 1-5, and loci included in such subsets are together, individually, and/or in randomly selected subsets, at least as informative (e.g., as statistically significant and/or reliable) for uses disclosed herein, e.g., for determining PSMA expression level.
  • a sample or subject from which the sample is obtained or derived is determined to have a particular PSMA expression level if 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more H3K27ac analyte loci identified in Table 4 are differentially H3K27ac modified as compared to a reference (e.g., a sample from (i) a healthy subject or cohort of healthy subjects or (ii) a subject with aberrant PSMA expression or a cohort of subjects with aberrant PSMA expression (e.g., a subject or cohort of subjects with mCRPC and that exhibit a positive PSMO PET SUVmean signal)).
  • a reference e.g., a sample from (i) a healthy subject or cohort of healthy subjects or (ii) a subject with aberrant PSMA expression or a cohort of subjects with aberrant PSMA expression (e.g., a subject or cohort of subjects with mCRPC and that exhibit a positive PSMO PET SUVmean signal)).
  • a sample or subject from which the sample is obtained or derived is determined to have a particular PSMA expression level if the H3K27ac analyte locus identified in Table 5 is differentially H3K27ac modified as compared to a reference (e.g., a sample from (i) a healthy subject or cohort of healthy subjects or (ii) a subject with aberrant PSMA expression or a cohort of subjects with aberrant PSMA expression (e.g., a subject or cohort of subjects with mCRPC and that exhibit a positive PSMO PET SUVmean signal)).
  • a reference e.g., a sample from (i) a healthy subject or cohort of healthy subjects or (ii) a subject with aberrant PSMA expression or a cohort of subjects with aberrant PSMA expression (e.g., a subject or cohort of subjects with mCRPC and that exhibit a positive PSMO PET SUVmean signal)).
  • a sample or subject from which the sample is derived is determined to have a particular PSMA expression level if one or more enhancer regions of one or more H3K27ac analyte genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) in Table 1 are differentially H3K27ac modified as compared to a reference (e.g., a sample from (i) a healthy subject or cohort of healthy subjects or (ii) a subject with aberrant PSMA expression or a cohort of subjects with aberrant PSMA expression (e.g., a subject or cohort of subjects with mCRPC and that exhibit a positive PSMO PET SUVmean signal)).
  • a reference e.g., a sample from (i) a healthy subject or cohort of healthy subjects or (ii) a subject with aberrant PSMA expression or a cohort of subjects with aberrant PSMA expression (e.g., a subject or cohort of subjects with mCRPC and that exhibit a positive PSMO PET SUVmean signal)).
  • differentially H3K27ac modified refers to an acetylation status characterized by an increase or decrease in a value measuring acetylation (e.g., of read counts and/or normalized read counts for a given genomic locus), and/or a mean, median and/or mode thereof, and/or a log thereof (e.g., log base 2 (log2)), of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40- fold, 45-fold, 50-fold, or greater, or any range in between, inclusive, such as 1% to 50%, 50% to 2-fold, 25% to 50-fold, 25% to 30-fold, 25% to
  • an increase or decrease in a value measuring acetylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or greater, or any range in between, inclusive, such as an increase or decrease of 0.1-fold to 10- fold, 0.2-fold to 5-fold, 0.2-fold to 4.0-fold, 0.4-4.0-fold, 0.4-fold to 4.0-fold, 0.6-fold to 4.0- fold, 0.8-fold to 4.0-fold, 1.0-fold to 4.0-fold.
  • a log2(fold-change) e.g., a log2(fold-
  • one or more enhancer regions of a recited gene are provided in Tables 1 and 2.
  • one or more enhancer regions of a recited gene corresponds to: (i) one or more loci with increased or decreased H3K27ac modifications as compared to a reference (e.g., a sample from a healthy subject) within a certain number of nucleotides (e.g., 50,000 nucleotides) of the recited gene; and/or (ii) one or more loci with increased or decreased H3K27ac modifications as compared to a reference (e.g., a sample from a healthy subject) that are closest to the recited gene in the genome.
  • a reference e.g., a sample from a healthy subject
  • Exemplary genomic loci whose DNA methylated state is associated with PSMA expression level are provided in Table 1 (see MBD analyte loci).
  • a person of skill in the art will recognize that the methods disclosed herein do not require that every MBD analyte genomic locus listed in Table 1 be assessed for DNA methylation. Instead, a subset of MBD loci may be assessed for DNA methylation. Subsets of the MBD genomic loci of Table 1 can be selected (e.g., for use in determining PSMA expression level) based on various performance criteria, e.g., to select genomic loci that demonstrate differential modification with a particular level of statistical significance and/or a particular threshold of differential between relevant states (e.g., a measured log2(fold-change)). Subsets of the genomic loci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier.
  • subsets of loci of Table 1 are together, individually, and/or in randomly selected subsets, at least as informative (e.g., as statistically significant and/or reliable) for uses disclosed herein, e.g., for determining PSMA expression level.
  • a sample or subject from which the sample is derived is determined to have a particular PSMA expression level if one or more MBD analyte loci (e g., 1,
  • a sample or subject from which the sample is obtained or derived is determined to have a particular PSMA expression level if DNA methylation for 1, 2,
  • 3, 4, 5, 6, 7, or 8 of the MBD analyte loci that are identified in Table 1 as having a positive association with PSMA expression are increased relative to a reference (e.g., a sample from (i) a healthy subject or cohort of healthy subjects or (ii) a subject with aberrant PSMA expression or a cohort of subjects with aberrant PSMA expression (e g., a subject or cohort of subjects with mCRPC and that exhibit a positive PSMO PET SUVmean signal)).
  • a reference e.g., a sample from (i) a healthy subject or cohort of healthy subjects or (ii) a subject with aberrant PSMA expression or a cohort of subjects with aberrant PSMA expression (e g., a subject or cohort of subjects with mCRPC and that exhibit a positive PSMO PET SUVmean signal)).
  • Genomic loci provided in Tables 1-2 can also demonstrate differential chromatin accessibility or transcription factor binding in different PSMA expression states.
  • histone methylation corresponds and/or is correlated with chromatin accessibility.
  • histone acetylation corresponds and/or is correlated with chromatin accessibility.
  • DNA methylation corresponds and/or is correlated with chromatin accessibility.
  • chromatin accessibility corresponds and/or is correlated with H3K4me3 modifications.
  • PSMA expression level may be determined by detecting and quantifying chromatin accessibility at one or more genomic loci in Tables 1 and 2 in accordance with the section above discussing exemplary genomic loci with differential H3K4me3 modifications.
  • chromatin accessibility corresponds and/or is correlated with H3K27ac modifications.
  • PSMA expression level may be determined by detecting and quantifying chromatin accessibility at one or more genomic loci in Tables 1 and 2 in accordance with the section above discussing exemplary genomic loci with differential H3K27ac modifications.
  • chromatin accessibility corresponds and/or is correlated with DNA methylation.
  • PSMA expression can be measured by detecting and quantifying chromatin accessibility at one or more genomic loci in Tables 1 and 2 in accordance with the section above discussing exemplary genomic loci with differential DNA methylation.
  • histone methylation corresponds and/or is correlated with transcription factor binding.
  • histone acetylation corresponds and/or is correlated with transcription factor binding.
  • DNA methylation corresponds and/or is correlated with transcription factor binding.
  • Methods, kits and systems of the present disclosure include analysis of differentially modified and/or differentially accessible genomic loci to measure disease-specific PSMA expression. Methods, kits and systems of the present disclosure can be used in any of a variety of applications. For example, methods, kits and systems of the present disclosure can be used in detecting and/or treating a disease or indication that can be associated with increased disease-specific PSMA expression (e.g., mCRPC). Methods, kits and systems of the present disclosure can also be used to detect or determine resistance of a disease or condition to a certain therapeutic (e.g., a PSMA-targeted therapeutic).
  • a disease or indication e.g., mCRPC
  • Methods, kits and systems of the present disclosure can also be used to detect or determine resistance of a disease or condition to a certain therapeutic (e.g., a PSMA-targeted therapeutic).
  • methods, kits and systems of the present disclosure can be applied to an asymptomatic human subject.
  • a subject can be referred to as “asymptomatic” if the subject does not report, and/or demonstrate by non-invasively observable indicia (e.g., without one, several, or all of device-based probing, tissue sample analysis, bodily fluid analysis, surgery, or autoimmune screening), sufficient characteristics of a disease or condition that can be associated with increased PSMA expression to support a medically reasonable suspicion that the subject is likely suffering from a disease or condition that can be associated with increased PSMA expression. Detection of early-stage diseases or conditions that can be associated with increased PSMA expression can be achieved using methods, kits and systems of the present disclosure, with attendant medical benefits including potential for early treatment and attendant improvement in therapeutic outcomes.
  • a subject can be referred to as “symptomatic” if the subject report, and/or demonstrates by non-invasively observable indicia (e.g., without one, several, or all of device-based probing, tissue sample analysis, bodily fluid analysis, surgery, or prostate cancer screening), sufficient characteristics of a disease or condition that can be associated with increased PSMA expression (including, e.g., prostate cancer (e.g., CRPC)) to support a medically reasonable suspicion that the subject is likely suffering from a disease or condition that can be associated with increased PSMA expression.
  • non-invasively observable indicia e.g., without one, several, or all of device-based probing, tissue sample analysis, bodily fluid analysis, surgery, or prostate cancer screening
  • sufficient characteristics of a disease or condition that can be associated with increased PSMA expression including, e.g., prostate cancer (e.g., CRPC)
  • methods, kits and systems of the present disclosure can be applied to a human subject previously determined to have a disease or condition that can be associated with increased PSMA expression.
  • methods, kits and systems of the present disclosure can be applied to a human subject previously determined to have prostate cancer (e.g., mCRPC)).
  • methods, kits and systems of the present disclosure can be used to determine that a subject has a PSMA expression level that correlates with a prior determination of PSMA expression level (e.g., based on imaging and/or one or more biomarkers). In some embodiments, methods, kits and systems of the present disclosure can be used to validate or confirm a prior determination that a subject has a certain PSMA expression level.
  • PSMA expression level improves diagnosis, prognosis, and treatment of a disease or indication that can be associated with increased PSMA expression, including and/or particularly an early stage disease or indication that can be associated with increased PSMA expression (e.g., mCRPC).
  • a disease or indication that can be associated with increased PSMA expression
  • an early stage disease or indication that can be associated with increased PSMA expression
  • the present disclosure provides, among other things, methods, kits and systems particularly useful for the diagnosis and treatment of early- stage diseases that can be associated with increased PSMA expression (e.g., mCRPC).
  • PSMA expression level determination in accordance with the present disclosure is performed once for a given subject or multiple times for a given subject.
  • PSMA expression level determination in accordance with the present disclosure is performed on a regular basis, e.g., every six months, annually, every two years, every three years, every four years, every five years, or every ten years.
  • methods, kits and systems disclosed herein provide a determination of PSMA expression level. In other instances, methods, kits and systems disclosed herein will be indicative of PSMA expression level but not definitive for PSMA expression level. In various instances in which methods, kits and systems of the present disclosure are used to determine PSMA expression level, the same can be followed by a further confirmatory assay, which further assay can confirm, support, undermine, or reject a determination resulting from a prior determination, e.g., a determination in accordance with the present disclosure. As used herein, a confirmatory assay can be a test that is currently recognized by medical practitioners, e.g., based on imaging or other testing.
  • PSMA expression level determination is followed by treatment with a PSMA- targeted therapeutic (e.g., 177Lu-PSMA-617).
  • a PSMA- targeted therapeutic e.g., 177Lu-PSMA-617.
  • treatment with a PSMA- targeted therapeutic includes administration of one or more therapies provided herein, including without limitation a radioligand conjugate and/or an ADC.
  • treatment of a disease or indication associated with increased PSMA expression includes administration of a therapeutic regimen including one or more treatments provided herein as available, appropriate, and/or preferred for a particular PSMA expression level.
  • methods, kits and systems can be used to determine whether a particular subject is likely to be and/or is characterized as responsive to a PSMA- targeted agent. In some such embodiments, methods, kits and systems can be followed by treatment of the subject with a PSMA-targeted agent.
  • methods, kits and systems can be used to determine whether a particular subject is likely to be and/or is characterized as resistant to, non-responsive to, or not recommended treatment with a PSMA-targeted agent. In some such embodiments, methods, kits and systems can be followed by treatment with a therapeutic agent to a different target.
  • Responsiveness can refer to the ability or likelihood of a therapy to cause a reduction in the number and/or size of tumor lesions, an increase in the time to next treatment, slowing of disease progression (e.g., as measured by plasma PSA levels for prostate cancer, and/or clinical or radiological evidence of progression), reduced disease activity, and/or increased survival.
  • Responsiveness can refer to improvement in prognosis.
  • Responsiveness can refer to achievement of a treatment benefit, including e.g., improvement in one or more symptoms of a disease or indication associated with increased PSMA expression.
  • Responsiveness can be measured quantitatively (e.g., as in the case of tumor size and/or number, PSA concentration, histone modification, chromatin accessibility, transcription factor binding, or DNA methylation at one or more genomic loci; or as in the calculation of clinical benefit (CBR)), or qualitatively (e.g., by measures such as “pathological complete response” (pCR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD), or other qualitative criteria).
  • CBR clinical benefit
  • Methods of the present disclosure can also be programmed or modeled in mathematical software packages that allow symbolic entry of equations and high-level specification of processing, including specific algorithms to be used, thereby freeing a user of the need to procedurally program individual equations and algorithms.
  • Such packages include, e.g., Matlab from Mathworks (Natick, MA), Mathematica from Wolfram Research (Champaign, IL), S-Plus from MathSoft (Seattle, WA), R from R Foundation for Statistical Computing (Vienna, Austria), Python from Python Software Foundation (Wilmington, DE), or Perl from Perl Foundation (Holland, MI).
  • a computer system comprises a database for storage of genomic locus modification status and/or accessibility status data.
  • a single learning statistical classifier system such as a classification tree (e. ., random forest) is used. In other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem.
  • Examples of learning statistical classifier systems include, but are not limited to, those described in the Examples and also those using inductive learning (e.g., decision/classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), Probably Approximately Correct (PAC) learning, connectionist learning (e.g, neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons such as multi-layer perceptrons, multilayer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc.), reinforcement learning (e.g, passive learning in a known environment such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement learning, etc ), and genetic algorithms and evolutionary programming.
  • inductive learning e.g., decision/classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.
  • PAC Probably Approximately Correct
  • connectionist learning e.g
  • methods of the present disclosure can include sending classification results to a medical practitioner, e.g., an oncologist.
  • a medical practitioner e.g., an oncologist.
  • the present disclosure includes methods where a therapeutic agent or regimen is administered to a subject based on PSMA expression level (e.g., disease specific PSMA expression level).
  • PSMA expression level e.g., disease specific PSMA expression level
  • the therapeutic agent or regimen provided herein will be available, appropriate, and/or preferred for a certain PSMA expression level.
  • those of skill in the art will be aware of recommended and/or governmentally approved formulations and/or dosages for various therapeutic agents provided herein.
  • compositions for delivery of one or more therapeutic agents to a subject include pharmaceutical compositions for delivery of one or more therapeutic agents to a subject.
  • a pharmaceutical composition may be in any form known in the art, including formulations for administration according to any route known in the art.
  • a suitable means of administration can be selected based on the age and condition of a subject.
  • composition forms of the present disclosure can include, e.g., liquid, semi-solid and solid dosage forms.
  • Pharmaceutical composition forms of the present disclosure can include, e.g., liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, and liposomes. Selection or use of any particular form may depend, in part, on the intended mode of administration and therapeutic application.
  • compositions can be formulated for administration by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection) or a non-parenteral mode.
  • parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection or infusion.
  • a pharmaceutical composition of the present disclosure can be in an injectable or infusible form.
  • the present disclosure includes sterile formulations for injection or infusion, which can be formulated in accordance with conventional pharmaceutical practices.
  • sterile powders for the preparation of sterile injectable solutions methods for preparation include vacuum drying and freeze-drying that yield a powder of a composition described herein plus any additional desired ingredient (see below) from a previously sterile-fdtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition a reagent that delays absorption, for example, monostearate salts, and gelatin.
  • Route of administration can be parenteral, for example, administration by injection.
  • Administration by injection can be by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection.
  • Administration can be systemic or local.
  • a composition described herein can be therapeutically delivered to a subject by way of local administration.
  • local administration or “local delivery,” can refer to delivery that does not rely upon transport of the composition or therapeutic agent to its intended target tissue or site via the vascular system.
  • the composition may be delivered by injection or implantation of the composition or therapeutic agent or by injection or implantation of a device containing the composition or therapeutic agent.
  • the composition or therapeutic agent, or one or more components thereof may diffuse to an intended target tissue or site that is not the site of administration.
  • a pharmaceutical composition can be administered parenterally in the form of an injectable formulation comprising a sterile solution or suspension in water or another pharmaceutically acceptable liquid.
  • a pharmaceutical composition can be formulated by suitably combining the therapeutic molecule with pharmaceutically acceptable vehicles or media, such as sterile water and physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, binder, followed by mixing in a unit dose form required for generally accepted pharmaceutical practices.
  • pharmaceutically acceptable vehicles or media such as sterile water and physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, binder, followed by mixing in a unit dose form required for generally accepted pharmaceutical practices.
  • examples of oily liquid include sesame oil and soybean oil, and it may be combined with benzyl benzoate or benzyl alcohol as a solubilizing agent.
  • administration of a therapeutic agent as described herein is achieved by administering to a subject a nucleic acid encoding a therapeutic agent described herein.
  • Nucleic acids encoding a therapeutic agent described herein can be incorporated into a gene construct to be used as a part of a gene therapy protocol to deliver nucleic acids that can be used to express and produce therapeutic agent within cells.
  • Expression constructs of such components may be administered in any therapeutically effective carrier, e.g., any formulation or composition capable of effectively delivering the component gene to cells in vivo.
  • a pharmaceutical composition can include a therapeutically effective amount of a therapeutic agent described herein. Such effective amounts can be readily determined by one of ordinary skill in the art. A therapeutically effective amount can be an amount at which any toxic or detrimental effects of the composition are outweighed by therapeutically beneficial effects. In some embodiments, a dose can also be chosen to reduce or avoid production of antibodies or other host immune responses against a therapeutic agent. Those of skill in the art will appreciate that data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. In various embodiments, the amount of active ingredient included in a pharmaceutical composition is such that a suitable dose within the designated range can be administered to subjects. The dose and method of administration can vary depending on weight, age, condition, and other characteristics of a patient, and can be suitably selected as needed by those skilled in the art.
  • compositions including certain therapeutic agents can be administered as a fixed dose, or in a milligram per kilogram (mg/kg) dose.
  • an exemplary single dose of certain pharmaceutical compositions described herein can include certain therapeutic agents as described herein in an amount equal to, e.g., 0.001 to 1000 mg/kg, 1-1000 mg/kg, 1-100 mg/kg, 0.5-50 mg/kg, 0.1-100 mg/kg, 0.5-25 mg/kg, 1-20 mg/kg, and 1-10 mg/kg body weight.
  • Exemplary dosages of a composition described herein include, without limitation, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 4 mg/kg, 8 mg/kg, or 20 mg/kg. The present disclosure is not limited to such ranges or dosages.
  • the present disclosure further includes methods of preparing pharmaceutical compositions of the present disclosure and kits including pharmaceutical compositions of the present disclosure.
  • therapeutic agents of the present disclosure can be administered to a subject in a course of treatment that further includes administration of one or more additional therapeutic agents or therapies that are not therapeutic agents (e.g., surgery or radiation).
  • Combination therapies of the present disclosure can include simultaneous exposure of a subject to therapeutic agents of two or more therapeutic regimens.
  • an additional therapeutic agent or therapy administered in combination with a therapeutic agent as described herein can be administered such that administration of the therapeutic agent and the additional therapeutic agent or therapy are separated by one or more hours before or after, one or more days before or after, one or more weeks before or after, or one or more months before or after administration of the therapeutic agent.
  • the administration frequency and/or dosage of one or more additional therapeutic agents can be the same as, similar to, or different from the administration frequency of a therapeutic agent.
  • administration of a therapeutic agent can be to a subject having previously received, scheduled to receive, or in the course of a treatment regimen including an additional cancer therapy (e.g., prostate cancer therapy).
  • Administration of a therapeutic agent can, in some instances, improve delivery or efficacy of another therapeutic agent or therapy with which it is administered in combination.
  • therapeutic agent combination therapies can demonstrate synergy and/or greater-than-additive effects between a therapeutic agent and one or more additional therapeutic agents with which it is administered in combination.
  • a therapeutic agent can be administered in any effective amount as determined independently or as determined by the joint action of therapeutic agent and any of one or more additional therapeutic agents or therapies administered.
  • Administration of the therapeutic agent may, in some embodiments, reduce the therapeutically effective dosage, required dosage, or administered dosage of the additional therapeutic agent or therapy relative to a reference regimen for administration of additional therapeutic agent or therapy or therapy absent the therapeutic agent.
  • a composition described herein can replace or augment other previously or currently administered therapy. For example, upon treating with therapeutic agent, administration of one or more additional therapeutic agents or therapies can cease or diminish, e.g., be administered at lower levels.
  • kits for detecting modification and/or accessibility of one or more genomic loci include kits for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci.
  • Kits of the present disclosure can include, e g., reagents such as buffers and/or antibodies useful in the detection and quantification of histone modifications.
  • a kit of the present disclosure can include at least one antibody that selective binds a histone modification selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4mel, H3K4me2, or H3K4me3, or pan acetylation.
  • a kit of the present disclosure can include at least one antibody that selective binds H3K4me3 modifications.
  • a kit of the present disclosure can include at least one antibody that selective binds H3K27ac modifications.
  • a kit of the present disclosure can include instructional materials disclosing or describing the use of the kit in a method of measuring PSMA expression and/or treatment disclosed herein.
  • the kit comprises reagents for measuring chromatin accessibility via an ATAC-seq assay.
  • the system comprises reagents for quantifying H3K4me3 modifications for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the H3K4me3 analyte genomic loci in Table 1 and 2.
  • the system comprises reagents for quantifying H3K27ac modifications for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 H3K27ac analyte loci in Tables 1 and 2.
  • the system comprises reagents for quantifying H3K27ac modifications for 1, 2, 3, 4, 5, 6, 7, or 8 H3K27ac genomic loci in Table 2 and/or H3K4me3 modifications for the H3K4me3 genomic loci in Table 2.
  • the system comprises one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones.
  • the system comprises reagents for quantifying H3K4me3 modifications for 1, 2, 3, or 4 of the H3K4me3 analyte genomic loci in Table 3.
  • the system comprises reagents for quantifying H3K4me3 modifications for one or both of the H3K4me3 analyte genomic loci in Table 4.
  • the system comprises reagents for quantifying H3K4me3 modifications for the H3K4me3 analyte genomic locus in Table 4.
  • the system comprises reagents for quantifying H3K27ac modifications for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 of the H3K27ac analyte genomic loci in Table 4. In some embodiments, the system comprises reagents for quantifying H3K27ac modifications for the H3K27ac analyte genomic locus in Table 4.
  • the system comprises reagents for quantifying DNA methylation for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the MBD analyte genomic loci in Table 1.
  • the system comprises one or more methyl-binding domains (e.g., for use in MBD-seq).
  • the system comprises one or more antibodies that can bind methylated DNA (e.g., for use in MeDIP).
  • the system comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample.
  • the sequencer comprises reagents for library preparation for sequencing.
  • the sequencer comprises reagents for sequencing.
  • the system comprises instructions for determining PSMA expression level.
  • the system comprises reagents for measuring chromatin accessibility via an ATAC-seq assay.
  • Accessibility Status or “Chromatin Accessibility Status”: As used herein, “accessibility status” or “chromatin accessibility status” of a genomic locus refers to the frequency with which DNA sequences corresponding to the genomic locus are identified in an assay for detection of accessible chromatin. Accessibility status can be determined by various assays known in the art, including without limitation ChlP-seq as one example. Where two samples are separately analyzed by the same assay or comparable assays for detection of accessible DNA sequences, differences in chromatin accessibility status of genomic loci can be detected. Accessibility status can be compared to a standard or reference. A sample that has an accessibility status that differs in accessibility status from a standard or reference can be referred to as differentially modified.
  • Suitable assays for determining chromatin accessibility are known in the art.
  • Exemplary assays include ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing), Mnase-seq (Micrococcal Nuclease digestion with sequencing), Dnase hypersensitivity assay, and/or a fragmentomics assay.
  • ATAC-seq Assay of Transpose Accessible Chromatin sequencing
  • NOMe-seq Nucleosome Occupancy and Methylome sequencing
  • FAIRE-seq Formmaldehyde-Assisted Isolation of Regulatory Elements sequencing
  • Mnase-seq Merococcal Nuclease digestion with sequencing
  • Dnase hypersensitivity assay and/or a fragmentomics assay
  • the term “administration” typically refers to the administration of a disease appropriate (e.g., prostate cancer appropriate) treatment.
  • the disease appropriate treatment may comprise administering a composition to a subject, for example to achieve delivery of an agent that is, is included in, or is otherwise delivered by, the composition.
  • the disease appropriate treatment may comprise administering an appropriate surgical procedure or radiological procedure, optionally in combination with administration of a composition.
  • agent may refer to any chemical or physical entity, including without limitation any of one or more of an atom, e.g., a radioactive atom, molecule, compound, conjugate, polypeptide, polynucleotide, polysaccharide, lipid, cell, or combination or complex thereof.
  • an atom e.g., a radioactive atom, molecule, compound, conjugate, polypeptide, polynucleotide, polysaccharide, lipid, cell, or combination or complex thereof.
  • each heavy chain includes a heavy chain variable domain (VH) and a heavy chain constant domain (CH).
  • VH heavy chain variable domain
  • CH heavy chain constant domain
  • the heavy chain constant domain includes three CH domains: CHI, CH2 and CH3.
  • the “hinge” connects CH2 and CH3 domains to the rest of the immunoglobulin.
  • Each light chain includes a light chain variable domain (VL) and a light chain constant domain (CL), separated from one another by another “switch.”
  • Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4).
  • CDR1, CDR2, and CDR3 Complement determining regions
  • FR1, FR2, FR3, and FR4 four somewhat invariant “framework” regions
  • the three CDRs and four FRs are arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of a heavy and/or a light chain are typically understood to provide a binding moiety that can interact with an antigen.
  • an antibody is a polyclonal, monoclonal, monospecific, or multispecific antibody (e.g., a bispecific antibody).
  • an antibody includes at least one light chain monomer or dimer, at least one heavy chain monomer or dimer, at least one heavy chain-light chain dimer, or a tetramer that includes two heavy chain monomers and two light chain monomers.
  • antibody can include (unless otherwise stated or clear from context) any art-known constructs or formats utilizing antibody structural and/or functional features including without limitation intrabodies, domain antibodies, antibody mimetics, Zybodies®, Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, isolated CDRs or sets thereof, single chain antibodies, single-chain Fvs (scFvs), disulfide-linked Fvs (sdFv), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), cameloid antibodies, camelized antibodies, masked antibodies (e.g., Probodies®), affybodies, anti -idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), Small Modular ImmunoPharmaceuticals (SMIPs), single chain or Tandem diabodies (TandAb®), VHH
  • an antibody includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR) or variable domain.
  • an antibody can be a covalently modified (“conjugated”) antibody (e.g., an antibody that includes a polypeptide including one or more canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen, where the polypeptide is covalently linked with one or more of a therapeutic agent, a detectable moiety, another polypeptide, a glycan, or a polyethylene glycol molecule).
  • conjugated antibody e.g., an antibody that includes a polypeptide including one or more canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen, where the polypeptide is covalently linked with one or more of a therapeutic agent, a detectable moiety, another polypeptide, a glycan, or a polyethylene glycol molecule.
  • antibody sequence elements are humanized, primatized, chimeric, etc.,
  • An antibody including a heavy chain constant domain can be, without limitation, an antibody of any known class, including but not limited to, IgA, secretory IgA, IgG, IgE and IgM, based on heavy chain constant domain amino acid sequence (e.g., alpha (a), delta (8), epsilon (s), gamma (y) and mu (p)).
  • IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.
  • “Isotype” refers to the Ab class or subclass (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes.
  • a “light chain” can be of a distinct type, e.g., kappa (K) or lambda (X), based on the amino acid sequence of the light chain constant domain.
  • an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human immunoglobulins. Naturally produced immunoglobulins are glycosylated, typically on the CH2 domain. As is known in the art, affinity and/or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, antibodies produced and/or utilized in accordance with the present disclosure include glycosylated Fc domains, including Fc domains with modified or engineered glycosylation.
  • an antibody can be specific for a particular histone modification (e.g., an antibody can bind one histone modification, e.g., H3K27ac with a higher affinity than other histone modifications, under conditions that are commonly used in ChlP-seq experiments).
  • an antibody is specific for an H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4mel, H3K4me2, or H3K4me3 modification.
  • an antibody is specific for an H3K27ac modification.
  • an antibody is specific for an H3K4me3 modification.
  • an antibody is a “pan” antibody.
  • the term pan antibody refers to an antibody that can bind a group of histone modifications having one or more features that are similar.
  • a pan antibody is a pan-methylation antibody (e.g., an antibody that can bind a histone, e.g., H3 that comprises at least one methylated lysine, wherein the at least one methylated lysine can be at any one of a plurality of amino acid positions, e.g., in some embodiments, a pan-methylation antibody can bind an H3 protein comprising a methylated lysine at any position).
  • a pan antibody is a pan-acetylation antibody (e.g., an antibody that can bind a histone, e.g., H3 that comprises at least one acetylated lysine, wherein the at least one acetylated lysine can be at any one of a plurality of amino acid positions, e.g., a pan-acetylation antibody can bind an H3 protein comprising an acetylated lysine at any position).
  • a pan antibody can bind one or more histone modifications that are associated with transcription activation.
  • a pan antibody can bind one or more histone modifications that are associated with transcription silencing.
  • an “antibody fragment” refers to a portion of an antibody or antibody agent as described herein, and typically refers to a portion that includes an antigen-binding portion or variable region thereof.
  • An antibody fragment can be produced by any means. For example, in some embodiments, an antibody fragment can be enzymatically or chemically produced by fragmentation of an intact antibody or antibody agent. Alternatively, in some embodiments, an antibody fragment can be recombinantly produced, i.e., by expression of an engineered nucleic acid sequence. In some embodiments, an antibody fragment can be wholly or partially synthetically produced.
  • an antibody fragment (particularly an antigen-binding antibody fragment) can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more, in some embodiments at least about 200 amino acids.
  • Two events or entities are “associated” with one another, as that term is used herein, if the presence, level and/or form of one is correlated with that of the other.
  • a particular entity e.g., an epigenetic profile comprising one or more histone modifications at a set of genomic loci, etc.
  • two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another.
  • two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non- covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, or a combination thereof.
  • biological sample typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell) of interest, as described herein.
  • a biological source is or includes an organism, such as a human subject.
  • a biological sample is or includes a biological tissue or fluid.
  • a biological sample can be or include cells, tissue, or bodily fluid.
  • Bodily fluids refer to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g., blood, serum, plasma, Cowper’s fluid or preejaculate fluid, chyle, chyme, stool, interstitial fluid, intracellular fluid, lymph, menses, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vitreous humor, vomit).
  • a biological sample can be or include blood, blood components, cell-free DNA (cfDNA), circulating-tumor DNA (ctDNA), ascites, biopsy samples, surgical specimens, cellcontaining body fluids, sputum, saliva, feces, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph, gynecological fluids, secretions, excretions, skin swabs, vaginal swabs, oral swabs, nasal swabs, washings or lavages such as a ductal lavages or bronchoalveolar lavages, aspirates, scrapings, or bone marrow.
  • cfDNA cell-free DNA
  • ctDNA circulating-tumor DNA
  • a biological sample is a liquid biopsy sample obtained from a bodily fluid.
  • a biological sample is or includes DNA obtained from a single subject or from a plurality of subjects.
  • a biological sample can be a “primary sample” obtained directly from a biological source or can be a “processed sample”, i.e., a sample that was derived from a primary sample, e.g., via dilution, purification, mixing with one or more reagents, or any other processing step(s) as described herein.
  • a biological sample can also be referred to as a “sample.”
  • Combination therapy refers to administration to a subject of two or more therapeutic agents or therapeutic regimens such that the two or more therapeutic agents or therapeutic regimens together treat a disease, condition, or disorder of the subject.
  • the two or more therapeutic agents or therapeutic regimens can be administered simultaneously, sequentially, or in overlapping dosing regimens.
  • combination therapy includes but does not require that the two therapeutic agents or therapeutic regimens be administered together in a single composition, nor at the same time.
  • corresponding to may be used to designate the position/identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition.
  • a monomeric residue in a polymer e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide
  • corresponding to a residue in an appropriate reference polymer.
  • Two sequences can be identified as corresponding if they are identical or if they share substantial identity, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more residues.
  • a nucleic acid sequence can correspond to a sequence that is identical or substantially identical (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the complement of the nucleic acid sequence, e.g., over a length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more nucleic acid residues.
  • diagnosis includes the act, process, and/or outcome of determining whether, and/or the qualitative of quantitative probability that, a subject has or will develop the condition, disease, or related state.
  • diagnosing can include a determination relating to prognosis and/or likely response to one or more general or particular therapeutic agents or regimens.
  • Expression level, amount, or level As used herein, the terms “expression level,” “amount,” or “level,” or used herein interchangeably, of a biomarker is a detectable level in a biological sample. “Expression” generally refers to the process by which information (e.g., gene-encoded and/or epigenetic) is converted into the structures present and operating in the cell. Therefore, as used herein, “expression” may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide).
  • Expression level, amount, or level of a given polypeptide is a measure of the expression process for that polypeptide.
  • Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g., by proteolysis.
  • “Expressed genes” include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs). Expression levels can be measured by methods known to one skilled in the art and also disclosed herein.
  • the expression level or amount of a biomarker can be used to identify/characterize a subject having a prostate cancer (e.g., mCRPC) who may be likely to respond to, or benefit from, a particular therapy (e.g., a PSMA-targeted therapy).
  • the expression level or amount of a biomarker provided herein in a subject having a prostate cancer described herein can also be used to determine and/or track the benefit of an administered therapy over time.
  • Identity refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules) and/or between polypeptide molecules. Methods for the calculation of a percent identity as between two provided sequences are known in the art. The term “% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between protein and nucleic acid sequences as determined by the match between strings of such sequences. “Identity” (often referred to as “similarity”) can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M.
  • Methods to determine identity and similarity are codified in publicly available computer programs. For example, calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences (or the complement of one or both sequences) for optimal comparison purposes (e g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and nonidentical sequences can be disregarded for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position.
  • residue e.g., nucleotide or amino acid
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, optionally accounting for the number of gaps, and the length of each gap, which may need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a computational algorithm, such as BLAST (basic local alignment search tool). Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin).
  • GCG Genetics Computer Group
  • BLASTP BLASTN
  • BLASTX Altschul et al., J Mol Biol (1990) 215:403-410
  • DNASTAR DNASTAR, Inc., Madison, Wisconsin
  • FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput Methods Genome Res [Proc Int Symp] (1994), Meeting Date 1992, 111-120. Eds. Suhai, Sandor. Plenum, New York, NY (the contents of each of which is separately incorporated herein by reference in its entirety).
  • Modification Status or “Histone Modification Status” : As used herein, “modification status” or “histone modification status” of a genomic locus refers to the frequency with which DNA sequences corresponding to the genomic locus are identified in an assay for detection of DNA sequences associated with histones bearing one or more histone modifications (e g., one or more particular histone modifications) and/or the density (e g., the measured density) of histone modifications (e.g., one or more particular histone modifications) corresponding to the genomic locus. Modification status can be determined by various assays known in the art, including without limitation ChlP-seq as one example.
  • CUT&RUN Cleavage Under Targets and Release Using Nuclease
  • CUT&Tag Cleavage Under Targets and Tagmentation
  • Modification status can be compared to a standard or reference.
  • a sample that has a modification status that differs in modification status or histone modification status from a standard or reference can be referred to as differentially modified.
  • PSMA expression refers to the amount of PSMA produced in a subject and/or the amount of PSMA produced in a subset of cells within a subject.
  • the subset of cells includes or consists of cells from a particular organ or tissue type (e.g., prostate tissue) of interest.
  • the subset of cells includes or consists of diseased cells.
  • the subset of cells includes or consists of cancer cells.
  • the subset of cells includes or consists of prostate cancer cells.
  • the subset of cells includes or consists of mCRPC cells.
  • “disease specific PSMA expression” refers to PSMA produced by diseased cells.
  • PSMA expression refers to cell surface and/or extracellular expression.
  • PSMA expression measured using technologies described herein can be associated with estimates of PSMA expression determined using other methods.
  • technologies provided herein for measuring PSMA expression level can be used to predict values that would be provided by other technologies, including, e.g., measurements provided by other approaches that have been shown to be associated with clinical outcomes and/or be suitable for determining patient eligibility for a certain therapeutic.
  • technologies provided herein can be used to predict a PSMA expression level determined using an imaging method, including, e.g., a PSMA PET imaging method.
  • technologies provided herein can be used to predict a PSMA PET SUVmean measurement.
  • PSMA Targeted Therapeutic refers to a therapeutic or administration of a therapeutic that can bind to or associate with PSMA (e.g., bind to or associate with PSMA in a subject).
  • a PSMA targeted therapeutic comprises a moiety that can bind to or associate with PSMA in a subject.
  • a PSMA targeted therapeutic comprises an antibody moiety that can bind to or associate with PSMA in a subject.
  • a PSMA targeted therapeutic comprises a small molecule moiety that can bind to or associate with PSMA in a subject.
  • a PSMA targeted therapeutic is an ADC comprising an antibody moiety that can bind PSMA (e.g., an antibody moiety of an ADC described herein).
  • a PSMA targeted therapeutic is a radiolabeled conjugate.
  • promoter signal refers to an epigenetic modification in a promoter region that is associated with increased expression of a gene regulated by the promoter region.
  • promoter signals include, e.g., histone methylation (e.g., H3K4me3).
  • promoter signal can be measured by quantifying histone methylation (e.g., H3K4me3), chromatin accessibility, and/or transcription factor binding.
  • a regulatory sequence is a nucleic acid sequence that controls expression of a coding sequence, e.g., a promoter sequence or an enhancer sequence.
  • a regulatory sequence can control or impact one or more aspects of gene expression (e.g., celltype-specific expression, inducible expression, etc.).
  • Subject refers to an organism, typically a mammal (e.g., a human).
  • a subject is suffering from a disease, disorder or condition (e.g., mCRPC).
  • a subject is susceptible to a disease, disorder, or condition.
  • a subject displays one or more symptoms or characteristics of a disease, disorder or condition.
  • a subject is not suffering from a disease, disorder or condition.
  • a subject does not display any symptom or characteristic of a disease, disorder, or condition.
  • a subject has one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition.
  • a subject is a subject that has been tested for a disease, disorder, or condition, and/or to whom therapy has been administered.
  • a human subject can be interchangeably referred to as a “patient” or “individual”.
  • therapeutic agent refers to any agent that elicits a desired pharmacological effect when administered to a subject.
  • an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population.
  • the appropriate population can be a population of model organisms or a human population.
  • an appropriate population can be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc.
  • a therapeutic agent is a substance that can be used for treatment of a disease, disorder, or condition (e.g., mCRPC).
  • a therapeutic agent is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans.
  • a therapeutic agent is an agent for which a medical prescription is required for administration to humans.
  • therapeutically effective amount refers to an amount that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition (e.g., mCRPC) in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual.
  • treatment refers to administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, or condition, or is administered for the purpose of achieving any such result.
  • a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, or condition, or is administered for the purpose of achieving any such result.
  • such treatment can be of a subject who does not exhibit signs of the relevant disease, disorder, or condition and/or of a subject who exhibits only early signs of the disease, disorder, or condition (e g., mCRPC).
  • such treatment can be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
  • treatment can be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition.
  • treatment can be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, or condition.
  • a “prophylactic treatment” includes a treatment administered to a subject who does not display signs or symptoms of a condition to be treated or displays only early signs or symptoms of the condition to be treated such that treatment is administered for the purpose of diminishing, preventing, or decreasing the risk of developing the condition.
  • a prophylactic treatment functions as a preventive treatment against a condition.
  • a “therapeutic treatment” includes a treatment administered to a subject who displays symptoms or signs of a condition and is administered to the subject for the purpose of reducing the severity or progression of the condition.
  • a method of predicting tumor specific PSMA expression e.g., predicting PSMA expression measurements determined using (i) an imaging procedure, (ii) a radioligand, and/or (iii) PSMA PET imaging (e.g., PSMA PET SUVmax or PSMA PET SUVmean)) in a subject, the method comprising: quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) or ctDNA from a liquid biopsy sample, obtained or derived from the subject:
  • any one of embodiments 1-23, comprising quantifying one or more histone modifications and/or DNA methylation for one or more of AMN, ARHGEF37, C4orf36, CADM1, CCDC175, CDC7, CLSTN1, COL5A1, EDNRA, FOLH1, GALR3, MED13L, MICB, NDRG3, NEDD1, NPAS2, NPVF, OLFM1, PCBP4, PROZ, PRRG3, RREB1, SCUBE3, SERPINA5, SNRPF, SORCS3, ST8SIA5, TEX19, TMEM132B, or TTC29 or any combination thereof, or one or more regulatory regions of any one of the foregoing (e.g., one or more promoter and/or enhancer regions o AMN, ARHGEF37, C4orf36, CADM1, CCDC175, CDC7, CLSTN1, COL5A1, EDNRA, FOLH1, GALR3, MEDI3L, MICB, NDRG3,
  • promoter signal e.g., H3K4me3 modifications
  • H3K4me3 modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 H3K4me3 analyte genomic loci in Table 1;
  • promoter signal e.g., H3K4me3 modifications
  • H3K4me3 modifications for 1 or 2 H3K4me3 analyte genomic loci in Table 4;
  • enhancer signal e.g., H3K27ac modifications
  • H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 H3K27ac analyte genomic loci in Table 4;
  • promoter signal e.g., H3K4me3 modifications
  • enhancer signal e.g., H3K27ac modifications
  • promoter signal e g., H3K4me3 modifications
  • the subject has previously been diagnosed with a disease or condition that is associated with increased PSMA expression, optionally wherein the disease or condition that is associated with increased PSMA expression is prostate cancer; and/or
  • a method of measuring PSA expression in a subject comprising: quantifying, at one or more genomic loci in a biological sample, optionally in cell-free
  • cfDNA circulating tumor DNA
  • ctDNA circulating tumor DNA
  • a method of predicting PSA expression in a subject comprising: quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA) from a liquid biopsy sample, obtained or derived from the subject:
  • chromatin accessibility is quantified using a chromatin accessibility assay selected from ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde- Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), a DNase hypersensitivity assay, and a fragmentomics assay.
  • ATAC-seq Assay of Transpose Accessible Chromatin sequencing
  • NOMe-seq Nucleosome Occupancy and Methylome sequencing
  • FAIRE-seq Formmaldehyde- Assisted Isolation of Regulatory Elements sequencing
  • MNase-seq Merococcal Nuclease digestion with sequencing
  • DNase hypersensitivity assay a fragmentomics assay.
  • the transcription factor binding assay is selected from ChlP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.
  • promoter signal e.g., H3K4me3 modifications
  • liquid biopsy sample is a plasma sample, serum sample, or urine sample.
  • the prostate cancer is metastatic castration resistant prostate cancer (mCRPC);
  • prostate cancer is prostate adenocarcinoma (PRAD) or neuroendocrine prostate cancer (NEPC).
  • PRAD prostate adenocarcinoma
  • NEPC neuroendocrine prostate cancer
  • a method of identifying a subject with elevated PSMA expression comprising:
  • a method of identifying a subject with elevated PSA expression comprising:
  • PSA expression e.g., serum PSA, including, e.g., total PSA
  • a method of diagnosing a subject as having a disease or disorder associated with elevated PSMA expression comprising:
  • a method of diagnosing a subject as having a disease or disorder associated with elevated PSA expression comprising:
  • PSA expression e.g., serum PSA, including, e.g., total PSA
  • the reference is a predetermined threshold, a measurement from a liquid biopsy sample, a measurement from an imaging test, and/or a normalized value, optionally wherein the reference is a measurement from an imaging test (e.g., PSMA PET SUVmean) or sample obtained from (i) a healthy subject or a cohort of healthy subjects, or (ii) a subject or a cohort of subjects that have been diagnosed with a disease or disorder associated with increased PSMA and/or PSA expression.
  • an imaging test e.g., PSMA PET SUVmean
  • a method of prognosing a subject having a disease or disorder associated with increased PSMA expression comprising:
  • a method of prognosing a subject having a disease or disorder associated with increased PSA expression comprising:
  • PSA expression e.g., serum PSA
  • the reference is a predetermined threshold, a measurement from a liquid biopsy sample, a measurement from an imaging test (e.g., PSMA PET SUVmean), and/or a normalized value, optionally wherein the reference is a measurement from a liquid biopsy sample or imaging test (e.g., PSMA PET SUVmean) obtained from (i) a healthy subject or a cohort of healthy subjects, or (ii) a subject or a cohort of subjects that have been diagnosed with the disease or disorder associated with increased PSMA and/or PSA expression.
  • an imaging test e.g., PSMA PET SUVmean
  • a normalized value optionally wherein the reference is a measurement from a liquid biopsy sample or imaging test (e.g., PSMA PET SUVmean) obtained from (i) a healthy subject or a cohort of healthy subjects, or (ii) a subject or a cohort of subjects that have been diagnosed with the disease or disorder associated with increased PSMA and/or PSA expression.
  • the reference is the median, lower bound of the top tertile, or lower bound of the top quartile value of the PSMA expression level measured, tumor specific PSMA expression level (e.g., PSMA PET SUVmean value) predicted, or tumor specific PSMA expression level (e.g., PSMA PET SUVmean value) measured in the cohort of subjects having the disease or disorder associated with increased PSMA expression; and if the PSMA expression level or predicted tumor specific PSMA expression level in the subject is equal to or greater than the reference, the subject is predicted to have a higher-than- normal risk of experiencing worse than normal disease progression as measured by one or more clinical outcomes.
  • tumor specific PSMA expression level e.g., PSMA PET SUVmean value
  • tumor specific PSMA expression level e.g., PSMA PET SUVmean value
  • the PSMA PET SUVmean median is about 4 to about 8, about 5 to about 8, 5 to about 7, about 6 to about 9, about 5, about 6, about 7, or about 8;
  • the lower bound of the top tertile of PSMA PET SUVmean is about 6 to about 12, about 6 to about 10, about 8 to about 12, about 6, about 7, about 8, about 9, about 10, about 11, or about 12; or
  • the lower bound of the top quartile of PSMA PET SUVmean is about 6 to about 14, about 6 to about 12, about 8 to about 14, about 8, about 9, about 10, about 11, about 12, about 13, or about 14.
  • a measurement from an imaging test e.g., PSMA PET SUVmean
  • the measurement is a serum PSA measurement.
  • any one of embodiments 77-79, wherein the one or more clinical outcomes include (i) overall survival, (ii) time to next treatment, or (iii) progression free survival (e.g., as determined by PSA-PFS (plasma PSA levels) and/or crPFS (clinical or radiological evidence of progression).
  • the one or more clinical outcomes include (i) overall survival, (ii) time to next treatment, or (iii) progression free survival (e.g., as determined by PSA-PFS (plasma PSA levels) and/or crPFS (clinical or radiological evidence of progression).
  • a method of monitoring progression of a disease associated with elevated PSMA expression in a subject comprising, at a first and second point in time:
  • a method of monitoring progression of a disease associated with elevated PSA expression in a subject comprising, at a first and second point in time:
  • a method of treating a subject having a disease or disorder associated with increased PSMA expression comprising measuring PSMA expression or predicting tumor specific PSMA expression (e.g., PSMA PET SUVmean) in the subject using the method of any one of embodiments 1-33 or 63-67, and comparing the measured PSMA expression level or predicted tumor specific PSMA expression to a reference, and
  • PSMA expression or predicting tumor specific PSMA expression e.g., PSMA PET SUVmean
  • a method of identifying a subject having a disease or disorder associated with increased PSMA expression that is likely to respond to a therapeutic comprising measuring PSMA expression or predicting tumor specific PSMA expression (e.g., PSMA PET SUVmean) in the subject using the method of any one of embodiments 1-33 or 63-67, and comparing the measured PSMA expression level or predicted tumor specific PSMA expression to a reference, and
  • PSMA expression or predicting tumor specific PSMA expression e.g., PSMA PET SUVmean
  • a method of predicting the likelihood that a subject having a disease or disorder associated with increased PSMA expression will respond to a therapeutic comprising measuring PSMA expression or predicting tumor specific PSMA expression (e.g., PSMA PET SUVmean) in the subject using the method of any one of embodiments 1-33 or 63-67, and comparing the measured PSMA expression level or predicted tumor specific PSMA expression to a reference, wherein
  • tumor specific PSMA expression e.g., PSMA PET SUVmean
  • any one of embodiments 86-88 wherein the reference is a predetermined threshold, a measurement from a liquid biopsy sample, a measurement from an imaging test, and/or a normalized value, optionally wherein the reference is a measurement from (i) a healthy subject or a cohort of healthy subjects, or (ii) a subject or a cohort of subjects that have been diagnosed with the disease or disorder associated with increased PSMA expression.
  • any one of embodiments 86-89, wherein the reference is a PSMA expression level measured, a tumor specific PSMA expression level (e.g., PSMA PET SUVmean value) predicted, or a tumor specific PSMA expression level (e.g., PSMA PET SUVmean value) measured in a cohort of subjects having the disease or disorder associated with increased PSMA expression.
  • a tumor specific PSMA expression level e.g., PSMA PET SUVmean value
  • a tumor specific PSMA expression level e.g., PSMA PET SUVmean value
  • tumor specific PSMA expression level e.g., PSMA PET SUVmean value
  • tumor specific PSMA expression level e.g., PSMA PET SUVmean value
  • the PSMA PET SUVmean median is about 4 to about 8, about 5 to about 8, 5 to about 7, about 6 to about 9, about 5, about 6, about 7, or about 8;
  • the lower bound of the top tertile of PSMA PET SUVmean is about 6 to about 12, about 6 to about 10, about 8 to about 12, about 6, about 7, about 8, about 9, about 10, about 11, or about 12; or
  • the lower bound of the top quartile of PSMA PET SUVmean is about 6 to about 14, about 6 to about 12, about 8 to about 14, about 8, about 9, about 10, about 11, about 12, about 13, or about 14.
  • a method of treating a subject having a disease or disorder associated with increased PSA expression comprising measuring or predicting PSA expression (e.g., serum PSA, including, e.g., total serum PSA) in the subject using the method of any one of embodiments 34-67, and comparing the measured PSA or predicted PSA expression to a reference, and
  • PSA expression e.g., serum PSA, including, e.g., total serum PSA
  • a method of identifying a subject having a disease or disorder associated with increased PSA expression that is likely to respond to a therapeutic comprising measuring or predicting PSA expression (e.g., serum PSA, including, e.g., total PSA) in the subject using the method of any one of embodiments 33-67, and comparing the measured or predicted PSA expression to a reference, and
  • PSA expression e.g., serum PSA, including, e.g., total PSA
  • a method of predicting the likelihood that a subject having a disease or disorder associated with increased PSA expression will respond to a therapeutic comprising measuring or predicting PSA expression (e.g., serum PSA, including, e.g., total PSA) in the subject using the method of any one of embodiments 33-67, and comparing the measured or predicted PSA expression to a reference, wherein
  • PSA expression e.g., serum PSA, including, e.g., total PSA
  • the reference is a predetermined threshold, a measurement from a liquid biopsy sample, and/or a normalized value, optionally wherein the reference is a measurement from (i) a healthy subject or a cohort of healthy subjects, or (ii) a subject or a cohort of subjects that have been diagnosed with the disease or disorder associated with increased PSA expression.
  • the therapeutic is an ADC (e.g., PSMA- MMAE, MLN2704, ARX517), and/or wherein the therapeutic is a PSMA-targeted radionuclide (e.g., 177Lu-PSMA-617).
  • the therapeutic is 177Lu-PSMA-617.
  • 111 The method of any one of embodiments 68-110, wherein the subject has a plasma PSA concentration of 0-2000 ng/mL (e.g., at least about 4 ng/mL, at least about 10 ng/mL, 10-2000 ng/mL, 25-2000 ng/mL, 50-2000 ng/mL, 75-2000 ng/ML, 100-2000 ng/mL, 150-1000 ng/mL, 100-500 ng/mL, or 100-200 ng/mL), optionally wherein the plasma PSA concentration has been determined using the method of any one of embodiments 33-67.
  • 0-2000 ng/mL e.g., at least about 4 ng/mL, at least about 10 ng/mL, 10-2000 ng/mL, 25-2000 ng/mL, 50-2000 ng/mL, 75-2000 ng/ML, 100-2000 ng/mL, 150-1000 ng/mL, 100-500 ng/mL,
  • a kit comprising reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci, wherein the one or more genomic loci are selected from Tables 1-5.
  • kit of embodiment 112 wherein the kit comprises reagents for quantifying:
  • promoter signal e.g., H3K4me3 modifications
  • H3K4me3 modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 H3K4me3 analyte genomic loci in Table 1;
  • enhancer signal e.g., H3K27ac modifications
  • H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 H3K27ac analyte genomic loci in Table 1;
  • enhancer signal e.g., H3K27ac modifications
  • promoter signal e.g., H3K4me3 modifications
  • enhancer signal e.g., H3K27ac modifications
  • promoter signal e.g., H3K4me3 modifications
  • H3K4me3 modifications for 1 or 2 H3K4me3 analyte genomic loci in Table 4;
  • enhancer signal e.g., H3K27ac modifications
  • H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 H3K27ac analyte genomic loci in Table 4;
  • promoter signal e.g., H3K4me3 modifications
  • enhancer signal e.g., H3K27ac modifications
  • kit of embodiment 112 or 113 wherein the kit comprises one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones.
  • cfDNA cell-free DNA
  • ctDNA cell-free DNA
  • a disease or disorder associated with increased PSMA e.g., a cancer, prostate cancer, or mCRPC.
  • a computer system comprising a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform operations to perform the method of any one of embodiments 1-111.
  • a system for determining the disease or disorder status of a subject comprising a sequencer configured to generate a sequencing data set from a sample; and a non- transitory computer readable storage medium of embodiment 120 and/or a computer system of embodiment 121.
  • invention 122 or 123 further comprising a sample preparation device configured to prepare the sample for sequencing from a biological sample, optionally a liquid biopsy sample.
  • sample preparation device comprises reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci in cell- free DNA (cfDNA) or ctDNA from the biological sample, optionally the liquid biopsy sample.
  • promoter signal e.g., H3K4me3 modifications
  • H3K4me3 modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 H3K4me3 analyte genomic loci in Table 1;
  • enhancer signal e.g., H3K27ac modifications
  • H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 H3K27ac analyte genomic loci in Table 1;
  • enhancer signal e.g., H3K27ac modifications
  • promoter signal e.g., H3K4me3 modifications
  • enhancer signal e.g., H3K27ac modifications
  • promoter signal e.g., H3K4me3 modifications 1 or 2 H3K4me3 analyte genomic loci in Table 4;
  • enhancer signal e.g., H3K27ac modifications
  • H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 H3K27ac analyte genomic loci in Table 4;
  • promoter signal e.g., H3K4me3 modifications
  • enhancer signal e.g., H3K27ac modifications
  • reagents comprise one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones.
  • the device comprises reagents for isolation of cell-free DNA (cfDNA) or ctDNA from the biological sample, optionally the liquid biopsy sample.
  • cfDNA cell-free DNA
  • ctDNA cell-free DNA
  • the present Examples demonstrate the identification and use of differentially modified and/or differentially accessible genomic loci in cfDNA in plasma samples obtained from subjects with mCRPC. Loci identified in the present example can be useful, e.g., for detecting mCRPC, characterizing mCRPC disease severity, monitoring mCRPC, prognosing subjects with mCRPC, and informing patient treatment decisions.
  • Example 1 Materials and Methods
  • the present Example describes the materials and methods that were used to generate sequencing data that was then used in Examples 2 and 3 to identify differentially modified genomic loci and create models to predict PSMA PET SUVmean.
  • Plasma samples were prepared from whole blood collected in EDTA blood collection tubes or Streck cell-free DNA BCT with 4-6 hours of collection and plasma was stored at -80°C until use.
  • Whole blood was obtained from metastatic Castration-Resistant Prostate Cancer (mCRPC) patients under a protocol approved by an IRB. Patients had previously been determined to have mCRPC. Informed content was obtained in each case and samples were de-identified.
  • mCRPC metastatic Castration-Resistant Prostate Cancer
  • Chromatin immunoprecipitation (ChIP)
  • Chromatin immunoprecipitation (ChIP) for histone marks (H3K4me3 and H3K27ac) in plasma samples can be performed using methods similar to those previously described in Baca et al. “Liquid biopsy epigenomic profiling for cancer subtyping.” Nature medicine 29.11 (2023): 2737-2741, which is incorporated by reference herein in its entirety. Briefly, about 1 mL frozen plasma was thawed and then prepared for ChIP. The thawed plasma was incubated with antibodies that bind H3K4me3 modifications or H3K27ac modifications that were previously conjugated to magnetic epoxy beads (Invitrogen) with constant mild shaking overnight. The beads were then washed and rinsed. Sequencing libraries were generated from purified immunoprecipitated sample DNA and then sequenced.
  • Enrichment of DNA methylation was performed on DNA extracted from human plasma samples using the EpiMark® Methylated DNA Enrichment Kit (E2600S, available from New England Biolabs) following the manufacturer’s protocol. Briefly, cfDNA libraries were prepared and adaptors ligated. Then, the EpiMark® capture reagent was applied to each library sample following the manufacturer’s protocol. Enriched DNA libraries were amplified and sequenced.
  • ChlP-sequencing reads and MBD-sequencing reads were aligned to the human genome build hgl9 using the Burrows-Wheeler Aligner (BWA) version 0.7.15. Non-uniquely mapping and redundant reads were discarded.
  • BWA Burrows-Wheeler Aligner
  • FDR q- value
  • Example 2 Determination of Tumor PSMA Expression in Prostate Cancer From Blood Using a Novel Epigenomic Liquid Biopsy Platform
  • PSMA Prostate-Specific Membrane Antigen
  • Pluvicto 177Lu-PSMA-617
  • mCRPC metastatic castration-resistant prostate cancer
  • Treatment eligibility currently requires collecting a PSMA PET SUVmean measurement, a PET scan quantification of the level of PSMA positivity in tumor lesions throughout the body.
  • a PET scan quantification of the level of PSMA positivity in tumor lesions throughout the body.
  • therapeutic strategies in development targeting an array of cell surface proteins there is an emerging unmet need to quantify tumor drug target expression minimally invasively.
  • the present Example provides data demonstrating that technologies described herein can be used as an accurate, minimally invasive readout of tumor PSMA expression in mCRPC.
  • the present Example also demonstrates that technologies provided herein can be used to monitor the transcription state of tumor cells in a patient, and can also accurately predict PSMA PET SUVmean, which can be useful, e.g., for prognostic prediction and as a companion diagnostic for PSMA radioimmune conjugates (RICs) (e.g., RICs approved or in development).
  • RICs radioimmune conjugates
  • FIGs. 1(A) and 1(B) Schematics summarizing the method used to characterize the epigenome of patients in the present Example are provided in Figs. 1(A) and 1(B).
  • Plasma samples were collected for 50 men previously diagnosed with mCRPC.
  • PET images were collected for 29 of the 50 men at the time of plasma collection, and the PET images were analyzed to quantify PSMA PET SUVmean.
  • MACS2 (—nolambda) was used to determine regions of the genome enriched for epigenomic signal. Consensus peak maps were then created for each analyte by merging maps across all patients (requiring a region to be covered by at least 3 patients’ peak maps) and removing regions known to be artifactual/technical (ENCODE blacklist). For each analyte’s consensus peak map, tiling across their regions was then performed using a 500 bp window, with a 100 bp step. Tiles were then analyzed to identify tiles having high-confidence mCRPC signal. Finally, high-confidence tiles were merged based on genomic coordinate overlaps.
  • This set of merged tiles are hereafter referred to as the “peaks”. Fragments within the peak regions (above local background) were quantified, normalized for read-depth, log2 -transformed (with a pseudo count of 0.01, and quantile normalized). For model validation this process was performed in a leave-one-out cross-validation schema.
  • differential analysis identified a number of regions having changes in epigenetic modifications in mCRPC subjects as compared to healthy subjects, including 15,174 loci with increased enhancer signal, 10,121 loci with decreased enhancer signal, 10,804 loci with increased promoter signal, 9,518 loci with decreased promoter signal, 41,198 loci with increased DNA methylation, and 9,238 loci with decreased DNA methylation.
  • genes associated with differential modifications were multiple prostate-cancer specific signals, including H0XB13, KLK2, KLK3, and SPDEF.
  • the detection of epigenetic modifications associated with multiple prostate-cancer specific signals demonstrates that technologies provided herein can be used to identify biologically relevant changes in the epigenome, that are reflective of transcription activity in tumor cells in a subject.
  • Table 1 Genomic Loci With Highest Association to PSMA PET SUVmean for H3K27ac, H3K4me3, and DNAme.
  • enhancer signal at the FOLH1 locus was identified as being most highly associated with PSMA PET SUVmean.
  • Fig. 4 provides exemplary epigenomic maps for H3K4me3 (promoter), H3K27ac (enhancers), and methylated DNA (DNAme) at the FOLH1 locus in subjects with low PSMA PET signal (defined as being below the median SUVmean of subjects tested), high PSMA PET signal (defined as being above the median SUVmean of subjects tested), and healthy subjects.
  • Epigenetic signal at the loci listed in the Table 2 (below) were found to provide particularly robust mCRPC-specific signal.
  • Table 2 Loci with particularly robust mCRPC-specific signal.
  • Table C Characteristics of Training and Validation Cohorts: [0441] All prostate cancer specific regions of the genome were normalized, allowing for per-experiment normalization of tumor specific regions and aggregated (aggregation can be performed, e.g., using a weighted sum product).
  • Performance was assessed via Pearson correlation in both a leave-one-out (LOO) cross-validation (CV) setting within the training cohort, as well as the held-out validation cohort using a final model trained on all data from the training cohort.
  • LEO leave-one-out
  • CV cross-validation
  • Results from the training cohort LOOCV and validation cohort analysis are shown in Fig. 5.
  • a positive correlation was observed between predicted PSMA PET SUVmean values and observed PSMA PET SUVmean values, demonstrating that epigenomic cfDNA profiling can provide an accurate surrogate of tumor PSMA expression in men with mCRPC, and can be useful for, e.g., detecting prostate cancer, characterizing prostate cancer in a subject, monitoring progression of prostate cancer, informing treatment selection, and optimizing patient selection.
  • the present Example provides a set of loci and analytes that are particularly useful for predicting PSMA expression levels. Surprisingly, it was found that monitoring epigenetic modifications at one or more of the small set of loci identified in Table 2 provided superior PSMA PET SUVmean predictive ability as compared to use of genome-wide epigenetic signal. Moreover, the ability of an ML model with loci identified in the present Example to predict PSMA PET SUVmean is a significant advancement in the field, as loci previously identified in the region were not found to be PRAD-specific/robust (data not provided).
  • the present Example provides results from a study testing the correlation between a PSMA scoring algorithm described herein and patient health outcomes.
  • PSMA PET SUVmean values were predicted for 84 samples, include 72 samples from subjects administered Pluvicto. Samples were then screened for a ctDNA fraction of > 0.03, resulting in 45 samples. The 45 samples were then split into tertiles based on PSMA PET SUVmean prediction scores and a logrank test was performed for 5 time-to-event clinical outcomes: PSA-PFS, crPFS, Time to Next Treatment, and Overall Survival.
  • the present Example provides data showing that technologies described herein can be used to measure PSA serum levels using epigenetic modifications measured in plasma samples.
  • a model for predicting PSA expression was generated using prostate cancer tumor biopsies. For each biopsy, genome wide maps of H3K4me3 and H3K27ac modifications were obtained using ChlP-seq. PSA expression was also determined for each biopsy using RNA- seq data. Simulated plasma samples were then generated by serially diluting the tumor biopsy sequencing data in silico with sequencing data from healthy plasma samples to create samples with a range of ctDNA%. Regions proximal to KLK3 (i.e., within KLK3 and +/- 200 kB of the transcript encoding portion of KI.K3) JVCQ identified that had epigenetic modification signal that correlated with PSA expression. These regions were then used to build a model for predicting PSA expression. The loci used in the present Example to measure PSA expression are provided in Table 3, below.
  • the model for predicting PSA expression was then applied to plasma samples obtained from subjects with prostate cancer to predict PSA expression level, and these predicted expression values were compared to serum PSA measurements in matched samples. Results of this comparison are shown in Fig. 8. As shown, predicted PSA expression was shown to correlate with measured serum PSA, demonstrating that technologies described herein can be used to measure serum PSA.
  • Example 5 Further PSMA PET SUV mea n Predictions in Prostate Cancer Patients [0454] The present Example provides further data demonstrating that technologies provided herein can be used predict PSMA PET SUVmean.
  • a model for measuring PSMA expression was generated using publicly available prostate tumor biopsy H3K4me3, H3K27Ac, and RNA-seq data. For each biopsy, genome wide maps of H3K4me3 and H3K27ac, and DNAme modifications were obtained. PSMA expression was also measured for each biopsy using RNA-seq.
  • the biopsy generated model was then applied to plasma samples obtained from patients with prostate cancer to obtain predicted PSMA expression values. These predicted values were compared to measured PSMA PET SUVmean in the same patients. Results are shown in Fig. 9(A). As shown, PSMA expression predicted using a biopsy model was shown to correlate with PSMA PET SUVmean signal, demonstrating that technologies described herein can be used to predict PSMA PET SUV mean signal.
  • Fig. 9(B) provides another characterization of the model constructed using the approach described in Examples 2 and 3.
  • the model generated in Example 3 (trained using PSMA PET SUVmean and epigenetic modifications from patient plasma samples) provided a model with improved accuracy as compared to a model generated using biopsy data.
  • sequencing data from plasma samples of patients with prostate cancer were diluted in silica with sequencing data from healthy subjects to generate in silico plasma samples having a range of ctDNA% values.
  • PSMA PET SUVmean was then predicted for each in silico sample using the biopsy model and the model trained using PSMA PET SUVmean (described in Example 3) and Pearson’s coefficients were determined. Results are shown in Fig. 9(C).
  • both models showed a strong Spearman correlation with PSMA PET SUVmean signal at clinically relevant concentrations, with the PSMA PET SUVmean trained model providing higher Spearman correlation at ctDNA% higher than ⁇ 2%.
  • the present Example provides further regions proximal to FOLH1 with H3K4me3 and H3K27ac signal that correlate with PSMA expression.
  • promoter and enhancer signal were identified in plasma samples from patients with lung cancer (SCLC).
  • SCLC lung cancer
  • the loci identified in the present Example can be used to measure or predict PSMA expression in prostate cancer.
  • Plasma samples from cancer patients and healthy volunteers were collected from commercial biobanks and stored at -80°C until use. The percentage of ctDNA in the plasma samples from cancer patients was assessed using ichorCNA, which estimates the percentage of ctDNA in a sample probabilistically (see Adalsteinsson et al., Nat Commun (2017) 8(1): 1324, the entire contents of which are incorporated herein by reference). Plasma samples from cancer patients with at least 5.5% ctDNA were used in the present Example.
  • H3K4me3 To identify promoter (H3K4me3) epigenomic activation signals a. FOLHl, we defined a peak within +/- Ikb of the transcription site (TSS) of FOLH1. To identify enhancer (H3K27ac) epigenomic activation signals at FOLH1, we defined a peak within +/- lOkb of the transcription site (TSS) of FOLHl. As a control, H3K4me3 and H3K27ac epigenomic activation signals were measured at housekeeping genes using a similar approach.
  • a consensus map of all peaks was created by taking the union of all base pairs covered by any peak in any of the cancer patient or healthy volunteer plasma samples. This set of regions was then combined with the set of “regions of interest” defined above to produce a set of “enriched regions”. The number of sequencing fragments (reads) overlapping each enriched region (by at least 1 bp) were quantified for each analyte. Counts of reads in all enriched regions between experiments (any region called a peak in at least one sample) were quantile normalized together. Quantile normalized counts of reads in the regions of interest were corrected for local ChlP-seq background to improve signal-to-noise.
  • Promoter and enhancer epigenomic activation signals were ctDNA corrected independently.
  • ichorCNA estimated values for each sample and regressed the log of the normalized, corrected counts against logit-transformed estimated ctDNA% with standard linear regression, and then subtracted the estimated percent of each count due to ctDNA% based on its regression weight. Corrected enhancer and promoter counts were summed for F0LH1 to produce an integrated activation score. The mean and standard-deviation of the summed enhancer and promoter counts within the healthy volunteers were used to calculate a z- score for each patient sample, which was then logged and 0-1 scaled for the final activation score.
  • Genomic coordinates of an exemplary H3K4me3 peak and an exemplary H3K27ac peak are provided in Table 5.
  • Table 5 Exemplary genomic coordinates of H3K4me3 and H3K27ac peaks for FOLH1.
  • Figs. 10(A) and (B) shows a trend line and confidence intervals for promoter signal (H3K4me3) and enhancer signal (H3K27ac), respectively, based on ctDNA% for PSMA.
  • the present Example provides further clinical trial data demonstrating that technologies described herein can predict responsiveness to treatment with a PSMA-targeted therapy (Pluvicto in the present Example).
  • the present Example provides data demonstrating that technologies provided herein can predict clinicoradiographic (CR) PFS, which provides a holistic assessment of progression by a clinician based on the totality of clinical data (including radiographic results).
  • this metric may more accurately reflect response to treatment with a PSMA-targeted agent (compared to PSA- PFS, time to next treatment (TTNT), and/or overall survival (OS).
  • Fig. 11 shows a comparison of CR PFS outcomes for subjects in (i) the bottom and middle tertiles of PSMA PET SUVmean values as compared to (ii) subjects in the top tertile.
  • technologies described herein were shown to be strong predictors of responsiveness of a PSMA-targeted therapy.
  • ctDNA% was measured for each patient, and patients were split into tertiles on the basis of ctDNA%.
  • Outcomes for four clinical trial metrics (PSA, time to next treatment, CR PFS, and overall survival) were compared for patients having a ctDNA% in the top tertile vs. patients having a ctDNA% in the middle and bottom tertiles. Results are shown in Figs. 12(A)-(D). As shown, ctDNA% was predictive of each of the clinical trial outcomes tested.
  • Predicted PSMA PET SUVmean was also shown to predict patient response to treatment, (see, e.g., Fig. 11, showing an HR of 0.27 for predicted PSMA PET SUVmean). This data demonstrates that technologies described herein, which use epigenetic modifications to predict PSMA PET SUVmean, can provide a predictor of responsiveness to a PSMA-targeted agent.

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Abstract

La présente divulgation concerne, entre autres, des procédés, des kits et des systèmes pour mesurer l'expression de PSMA et de PSA chez un sujet. Dans divers modes de réalisation, la présente divulgation concerne l'utilisation d'une ou de plusieurs modifications d'histone, l'accessibilité de la chromatine, la liaison d'un ou de plusieurs facteurs de transcription, et/ou la méthylation de l'ADN qui sont associés à des niveaux d'expression de PSA et de PSMA. Dans certains modes de réalisation, des modifications différentielles et/ou une accessibilité différentielle sont détectées et quantifiées au niveau d'un ou de plusieurs loci génomiques d'un échantillon biologique, par exemple, dans de l'ADN libre circulant (ADNcf) ou de l'ADN tumoral circulant (ADNct) à partir d'un échantillon de biopsie liquide obtenu ou issu d'un sujet atteint d'un cancer de la prostate (par exemple, mCRPC). Dans divers modes de réalisation, un état déterminé est utile, par exemple, pour sélectionner un traitement pour le cancer de la prostate (par exemple, mCRPC) et/ou pour traiter celui-ci.
PCT/US2025/023339 2024-04-06 2025-04-05 Procédés, kits et systèmes de mesure de l'expression de psa et de psma et méthodes de traitement du cancer sur la base de ceux-ci Pending WO2025213150A1 (fr)

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