WO2019055829A1 - Procédés de détection de biomarqueurs du cancer - Google Patents

Procédés de détection de biomarqueurs du cancer Download PDF

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WO2019055829A1
WO2019055829A1 PCT/US2018/051150 US2018051150W WO2019055829A1 WO 2019055829 A1 WO2019055829 A1 WO 2019055829A1 US 2018051150 W US2018051150 W US 2018051150W WO 2019055829 A1 WO2019055829 A1 WO 2019055829A1
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mutation
hist1h3b
h3f3a
plasma
tumor
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Javad NAZARIAN
Eshini PANDITHARATNA
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Nazarian Javad
Panditharatna Eshini
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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
    • C12Q2600/158—Expression markers

Definitions

  • the disclosure relates generally to detecting mutations in cancers of patients receiving cancer therapy and more particularly to detecting genetic mutations in brain cancers, including pediatric brain cancers.
  • midline anatomical brain structures e.g. pons, thalamus, spinal cord
  • DIPG diffuse intrinsic pontine glioma
  • MLGs harbor mutations in genes encoding canonical histones H3.1 (HIST1H3B/C), H3.2
  • the disclosure provides new methods for diagnosing cancers, including pediatric brain tumors.
  • the disclosure provides a method of determining the efficacy of a cancer therapy, said method comprising:
  • step c) comprises performing a method selected from the group consisting of quantitative polymerase chain reaction (qPCR), quantitative real-time polymerase chain reaction (qRTPCR), digital droplet PCR, (ddPCR), sequencing, northern blotting, or Southern blotting.
  • qPCR quantitative polymerase chain reaction
  • qRTPCR quantitative real-time polymerase chain reaction
  • ddPCR digital droplet PCR
  • the disclosure provides a method of treating cancer in a subject in need thereof, said method comprising:
  • step a) comprises performing digital PCR or drop digital PCR (ddPCR).
  • the disclosure provides a method of detecting a mutation in one or more genes selected from the group consisting of H3F3A, HIST1H3B, HIST1H3C, HIST2H3C, ACVRl, PPMID, PIK3R1, PIK3CA, IDHl, and BRAF in a blood or plasma sample from a human subject between 1 and 18 years of age, said method comprising performing digital PCR or droplet digital PCR (ddPCR) on the blood or plasma sample to determine the presence of a mutation in one or more genes selected from the group consisting of H3F3A, HIST1H3B, HIST1H3C, HIST2H3Q ACVRl, PPMID, PIK3R1, PIK3CA, IDHl, and BRAF in a biological sample.
  • the subject has cancer.
  • the cancer is a brain tumor.
  • the brain tumor is a diffuse glioma or a diffuse intrinsic potine gliom
  • the biological sample is a fluid.
  • the biological fluid is blood, plasma, serum, CSF, or urine.
  • the cancer is a brain tumor. In one embodiment, the cancer is a pediatric brain tumor. In some embodiments, the brain tumor is a diffuse glioma or a diffuse intrinsic pontine glioma (DIPG).
  • DIPG diffuse intrinsic pontine glioma
  • one or more genes comprise H3F3A.
  • the mutation is a K27M mutation in H3F3A.
  • the method further comprises determining the presence of a mutation in at least one other gene selected from the group consisting of HIST1H3B, HIST1H3C, HIST2H3C, ACVRl, PPMID, PIK3R1, PIK3CA, IDHl, and BRAF.
  • the mutation is a K27M mutation in H3F3A.
  • the method further comprises determining the presence of a mutation in at least one other gene selected from the group consisting of HIST1H3B, ACVRl, PPMID, PIK3R1, and PIK3CA. In another aspect of this embodiment, of any of the above methods, the method further comprises determining the presence of a mutation in at least one other gene selected from the group consisting of HIST1H3B, ACVRl, PPMID, and PIK3R1.
  • the disclosure provides an oligonucleotide probe comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 19-36, wherein the probe further comprises a label.
  • the disclosure provides a composition comprising at least one oligonucleotide probe comprising one of SEQ ID NOs: 19-36, wherein the probe further comprises a label.
  • FIGS. 1A and IB provide an overview of biofluid specimens utilized for detection of ctDNA in diffuse midline glioma patients.
  • FIG. 1A provides demographic, clinical, and molecular information for patients analyzed herein. Genomic alterations were obtained by tumor sequencing data generated for the PNOC003 trial, biorepository sequencing project, or other clinical studies.
  • FIG. IB is a flow chart of biofluid samples analyzed herein. Patients were either enrolled in PNOC003 trial (DIPG) or Children's National (CN) brain tumor biorepository (MLG); *patients enrolled on 1339 to donate specimens at postmortem.
  • DIPG PNOC003 trial
  • CN Children's National
  • MLG brain tumor biorepository
  • FIGS. 2A-N are graphical representations of genomic DNA validation for specific detection of mutant and wild type alleles of genes encoding driver mutations in pedatric diffuse midline gliomas. Shown are representative ddPCR plots for mutation detection using tumor genomic DNA from seven MLG patients and two non-CNS diseased brain tissue controls.
  • FIGS. 2A-2G are from tumor tissue;
  • FIGS. 2H-2N are from two non-CNS diseased brain tissue controls.
  • FIG. 3A-G are graphical representations of results obtained when assessing the sensitivity of a ddPCR platform with pre-amplification quantities of 2500 pg, 250 pg, 25 pg, and 2.5 pg of input DNA Genomic DNA.
  • the DNA was obtained from frozen tumor tissue obtained at postmortem, from a H3.3 K27M mutant DIPG patient was diluted and used to assess for sensitivity.
  • FIGS. 3A-3C are samples assessed without pre-amplification;
  • FIGS. 3D-3G are from samples assessed with preamplification.
  • FIGS. 4A-4B are graphical representations assessing the specificity of s ddPCR platform by testing for H3.3 K27M in H3 wildtype DIPG tumor tissue and liquid bio me.
  • FIG. 4A shows matched tumor tissue and CSF collected at postmortem from the same H3 WT DIPG patient who was negative for H3F3A p.K27M.
  • FIG. 4B shows that plasma collected at initial diagnosis was negative for H3F3A p.K27M in three H3 wild type DIPG patients.
  • FIG. 5A-B show schematics of biofluid specimen processing for ddPCR analysis.
  • FIG. 5A shows that genomic DNA was isolated from tumor tissue and used for ddPCR, while plasma and CSF were frozen upon collection, processed for ctDNA isolation, preamplified, and analyzed by ddPCR.
  • FIG. 5B shows that circulating DNA isolated from biofluid specimens were analyzed in a three-step method by ddPCR, which includes dropletization (by a'Source' machine), PCR, followed by detection in the 'Sense' machine.
  • An example is shown for H3F3A p.K27M detection, where LNA probes detect a single nucleotide change differentiating mutant and wild type alleles during PCR.
  • FIGS. 6A-F are graphic and pictorial representations showing detection of an histone 3 p.K27M mutation (H3K27M) ctDNA in biofluids of patients diagnosed with midline glioma.
  • FIG. 6A shows detection of H3K27M ctDNA in CSF collected throughout a disease.
  • FIG. 6B shows significantly higher ctDNA MAFs for H3K27M were detected in CSF compared to plasma.
  • FIG. 6C shows higher H3K27M MAFs were present in CSF compared to plasma in the same patient.
  • FIG. 6D shows that plasma ctDNA collected at diagnosis in DIPG patients enrolled in the PNOC003 clinical trial analyzed for H3K27M.
  • FIG. 6E shows that detection of H3K27M plasma ctDNA at post radiation in two patients that lacked detection at diagnosis.
  • FIG. 6F shows an Increase in tumor volume by MRI at post radiation compared to diagnosis in patients shown in FIG. 6C.
  • FIGS. 7A-F show multiplexed detection of oncohistone and partner mutations in tumor and CSF of MLG patients. Genomic DNA and CSF ctDNA were analyzed from the same MLG patient for multiplexed detection of mutations.
  • FIGS. 8A-C show CSF collected from various neuroanatomical locations in MLGs. Bar graphs represent average for H3K27M MAF detection from technical duplicates, and error bars represent standard error of mean.
  • FIG. 8A shows ignificantly higher H3K27M MAF was detected in CSF collected from adjacent sites to tumor compared to distant in MLG patients.
  • FIG. 8B shows Cohort based analysis of CSF collected from DIPG patients from different neuroanatomical locations, indicating higher detection in locations closer to brainstem.
  • FIG. 8C shows matched analysis for CSF collected from lateral ventricles and lumbar spine at postmortem from the same DIPG patient, showing similar trends as in (FIG. 8A) and (FIG. 8B).
  • FIGS. 9A-D show detection of a novel histone 3 mutation in fluid present in a brainstem tumor cyst found in a DIPG patient at postmortem.
  • FIG. 9A is an image of a cyst (indicated by an arrow) in fresh tissue at postmortem.
  • FIG. 9B is an MRI image captured a month before postmortem indicating cyst in pontine tumor.
  • FIGS. 9C-9D show that higher MAF levels were found in cyst fluid (41%) compared to tumor tissue (34%), and CSF (38%) for H3F3A p.K27M.
  • FIGS 10A-B shows detection of H3.3 K27M ctDNA in CSF at postmortem in a DIPG patient that tested negative in CSF collected at diagnosis.
  • FIG. 10A shows detection of
  • FIG.10B shows the results of an MRI imaging at diagnosis and postmortem for the same patient CSF was analyzed for in FIG. 10A.
  • FIGS. 11A-F show that a temporal analysis of plasma ctDNA matches response to therapy in DIPG patients.
  • FIG. 11 A shows serial plasma ctDNA analysis of changes in H3K27M throughout course of treatment in patients that followed PNOC003 recommended therapy.
  • FIG. 1 IB shows dynamic changes in plasma ctDNA and MRI tumor measurements in one patient in response to therapy. FLAIR and Tl-weighted post- gadolinium MRI images prior to treatment demonstrating a prominent expansile pontine mass and an additional focus within right cerebellar hemisphere that demonstrates evidence of enhancement. After treatment, the pontine and right cerebellar hemisphere lesions decreased in size.
  • FIG. 11C shows fluctuations in plasma ctDNA and MRI tumor volumes during therapy.
  • FIG. 1 ID shows a significant decrease in plasma ctDNA and MRI tumor volumes in response to radiation therapy.
  • FIG. 1 IE shows a decrease in plasma ctDNA and MRI tumor burden from diagnosis to pre-cycle 3 of PNOC003 recommended
  • FIGS. 12A-12N show longitudinal changes in plasma ctDNA in association with MR imaging findings and clinical assessments.
  • Each line graph represents plasma ctDNA and MRI changes in an individual patient diagnosed with DIPG.
  • the red line depicts MRI tumor measurements and black line depicts changes in temporal plasma ctDNA. Error bars are standard error of mean for technical triplicates of plasma ctDNA assessed for MAF of H3K27M.
  • the colored legend at the bottom of each figure indicates the time point of plasma ctDNA and MRI assessment during course of disease: green for initial diagnosis/biopsy, grey for during therapy, red for tumor growth, and black for the end of therapy following tumor growth/progression.
  • FIG. 13A-C are histograms showing bio fluid ctDNA and tumor spread as assessed by MRI, genomic, and/or histological studies.
  • FIG. 13A shows tumor extension beyond site of primary tumor (pons or thalamus) as determined based on MRI obtained prior to postmortem, or molecular and/or histopathology of autopsied whole brain specimens.
  • CSF ctDNA was higher in 18 MLG patients with tumor extension as compared to three MLG patients without tumor spread. Tumor involvement beyond pons was assessed by MRI review of patients enrolled in PNOC003.
  • Plasma ctDNA and MRI collected from DIPG patients were analyzed (FIG. 13B) at initial diagnosis and (FIG. 13C) at time points during the course of disease (initial diagnosis, during therapy, and tumor growth). Unlike in CSF, ctDNA levels in plasma were not higher in DIPG patients with tumor spread.
  • circulating tumor DNA to monitor disease progression is non/minimally- invasive method that has been increasingly employed for disease monitoring in adult cancers including glioblastoma (GBM), melanoma, lung, breast, and colon cancersi3-n74 ,however such studies have not been applied to the pediatric population.
  • GBM glioblastoma
  • melanoma melanoma
  • lung breast
  • colon cancersi3-n74 colon cancersi3-n74
  • Described herein is a sensitive, specific, rapid and minimally-invasive method for tumor surveillance of pediatric MLGs by monitoring the liquid biome.
  • plasma collected through an ongoing clinical trial (PNOC003; NCT 2274987) conducted by the Pacific Pediatric Neuro-Oncology Consortium (PNOC)
  • PNOC003; NCT 2274987 conducted by the Pacific Pediatric Neuro-Oncology Consortium
  • MAF mutation allelic frequency
  • the disclosure provides a robust application of the use of ctDNA for tumor profiling, and assessment of tumor response to therapy in pediatric patients with MLGs. These results show the feasibility of incorporating liquid biopsy as a sensitive and minimally invasive tool to inform clinical management for children with MLGs
  • circulating tumor DNA to monitor disease progression is a noninvasive method that has been shown to be successful in adult glioblastoma, lung cancer, melanoma, breast cancer, and colon cancer (Tie et al. (2016) Sci. Transl. Med., 8(346):346ra92; Tsao et al. (2015) Sci. Rep., 5:11198, Garcia-Murillas et al. (2015) Sci Transl Med.,
  • ctDNA is detectable in less than 50% of primary brain tumors (Bettegowda et al. (2014) Sci Transl. Med., 6(224):224ra24) and that allelic frequency of H3F3A is low (Lewis et al. (2013) Science 340(6134): 857-61 ; Bender et al. (2013) Cancer Cell
  • ctDNA was molecularly characterized in cerebrospinal fluid (CSF) and plasma, and the clinical utility of ctDNA to monitor tumor burden in pediatric midline gliomas (MLGs) was assessed.
  • CSF cerebrospinal fluid
  • MLGs pediatric midline gliomas
  • methods are performed using highly sensitive PCR-based sequencing method, such as digital droplet PCR.
  • digital PGR refers to PCR wherein the sample is divided into discrete subunits prior to amplification by PGR.
  • the sample may be separated into thousands or millions of partitions, each containing either zero or one (or, at most, a few) template molecules.
  • Fluorescent probes may be used to identify amplified target DNA in the partitions.
  • Samples containing amplified product e.g., fluorescent
  • those without product e.g., no fluorescence
  • the ratio of positives to negatives in each sample can be used for quantification. For example, Poisson statistics can be used to determine the absolute template quantity without the need to consider the number of amplification cycles.
  • ddPCR droplet digital PCR
  • the PCR solution is divided into smaller reactions (e.g., picoliter sized) through a water oil emulsion technique, which are then made to ran PCR individually.
  • the PCR sample is partitioned into nanoliter-size samples and encapsulated in oil droplets, (see, e.g., Hinson et al. (2011) Anal. Chem., 83:8604-8610; Pinheiro et al. (2012) Anal. Chem.,
  • PCR process is well known in the art and includes, for example, methods described in US Patent No. 9,984,201, the contents of which are incorporated by reference herein in their entirety. These methods include, e.g., reverse transcription PCR, ligation mediated PCR, digital PCR (dPCR), or droplet digital PCR (ddPCR).
  • dPCR digital PCR
  • ddPCR droplet digital PCR
  • PCR is carried out as an automated process with a thermostable enzyme.
  • the temperature of the reaction mixture is cycled through a denaturing region, a primer annealing region, and an extension reaction region automatically.
  • Machines specifically adapted for this purpose are commercially available.
  • amplified sequences are also measured using invasive cleavage reactions such as the InvaderTM technology (Zou et al, 2010, Association of Clinical Chemistry (AACC) poster presentation on Jul. 28, 2010, "Sensitive Quantification of Methylated Markers with a Novel Methylation Specific Technology; and U.S. Pat. No. 7,011,944 (Prudent et al)).
  • InvaderTM technology Zaou et al, 2010, Association of Clinical Chemistry (AACC) poster presentation on Jul. 28, 2010, "Sensitive Quantification of Methylated Markers with a Novel Methylation Specific Technology; and U.S. Pat. No. 7,011,944 (Prudent et al)).
  • next generation sequencing technologies are widely available. Examples include the 454 Life Sciences platform (Roche, Branford, Conn.) (Margulies et al. 2005 Nature, 437, 376-380); Ulumina's Genome Analyzer, GoldenGate Methylation Assay, or Infinium
  • Methylation Assays i.e., Infinium HumanMethylation 27K BeadArray or VeraCode GoldenGate methylation array (Illumina, San Diego, Calif.; Bibkova et al, 2006, Genome Res. 16, 383-393; U.S. Pat. Nos. 6,306,597 and 7,598,035 (Macevicz); U.S. Pat. No. 7,232,656 (Balasubramanian et al.)); QX200.TM. Droplet DigitaLTM. PCR System from Bio-Rad; or DNA Sequencing by Ligation, SOLiD System (Applied Biosystems/Life Technologies; U.S. Pat. Nos. 6,797,470,
  • methods are performed using digital droplet PCR (ddPCR).
  • ddPCR digital droplet PCR
  • digital droplet PCR is one of the most sensitive methods compared to Sanger sequencing, quantitative PCR, and next generation sequencing techniques (Diaz et al. (2014) J. Clin. Oncol. 32(6):579-86; Baker, M. (2012) Nat Meth., 9:541-544).
  • the nucleic acids may be quantified by counting the sub- samples that contain PCR end-product (positive reactions) and the sub- samples containing no PCR end-product (negative reactions) taking into account the Poisson distribution.
  • Digital PCR dPCR
  • dPCR Digital PCR
  • Digital droplet PCR as used herein relates to a digital PCR method in which the initial sample is sub-divided into several droplets constituting the sub- samples.
  • ddPCR was used to detect major mutations in ctDNA of liquid biome specimens and to assess dynamic changes in ctDNA during a disease course.
  • H3 27M partners with at least one additional driver mutation in cell cycle regulatory or growth factor signaling pathways (Nikbakht et al. (2016) Nat. Commun., 7:11185).
  • the presence of H3K27M and partner mutations in ctDNA was identified from liquid biome of MLGs.
  • the amount of detectable ctDNA in CSF and plasma was assessed with regard to clinical variables such as response to therapy, MRI tumor measurements, stage of disease, tumor spread, overall survival, and neuroanatomieal location of biofluid collection.
  • CSF samples were collected at various stages of disease (upfront, recurrence, and postmortem) from pediatric high grade midline gliomas.
  • DIPG diffuse intrinsic pontine glioma
  • a liquid biopsy assay using a sensitive and robust digital droplet PGR system on CSF and plasma samples was developed.
  • Major DIPG driver mutations including H3F3A (e.g., p. 27M),
  • HIST1H3B (e.g., p. 27M), ACVR1 (e.g., p.G328V/p.R206H), PPM ID (e.g., p.E525X),
  • PI 3R1 e.g., p.K567E
  • PI 3CA e.g., p.H1047R
  • Samples can also be analyzed for IDH1 (e.g., p,R132H) and BRAF (e.g., p.V600E) mutations.
  • IDH1 e.g., p,R132H
  • BRAF e.g., p.V600E
  • H3K27M was detected in ctDNA of plasma samples in xenograft models of DIPG.
  • biofluids from patients and animal models e.g., urine, blood, serum, plasma, CSF
  • a liquid biopsy can complement or in some cases eliminate the need for biopsies and inform recurrence and tumor response to treatment.
  • the method comprises determining the presence of a mutation in histone H3 (e.g., histone H3.3) and administering a therapy (e.g., administering at least one therapeutic agent to the subject) to the subject if the mutation in histone H3 is present.
  • the method may further comprise the step of obtaining a biological sample from the subject.
  • the biological sample of the methods may be a fluid or liquid such as blood, CSF, plasma, or urine.
  • the biological sample is blood or plasma.
  • the therapy administered to the subject is an epigenetic regulating drug such as, without limitation, SAHA and panobiiiostat.
  • the cancer detected and treated by the instant methods may be a brain tumor. More specifically, the cancer may be a pediatric brain tumor. In a particular embodiment, the cancer is a glioma, particularly a diffuse glioma or a diffuse intrinsic pontine glioma (DIPG).
  • the methods of the instant invention may comprise detenrdning the presence of a mutation(s) in circulating tumor DNA (ctDNA). In a particular embodiment, determining of the presence of the mutation(s) comprises perforating digital PCR. In a particular
  • the determining of the presence of the mutation comprises performing droplet digital PCR (ddPCR).
  • the digital PCR or ddPCR may be performed with at least one primer comprising any one of SEQ ID NOs: 1-18.
  • the digital PCR or ddPCR may be performed with primers comprising each of SEQ ID NOs: 1-18.
  • the digital PCR or ddPCR may be performed with at least one probe comprising any one of SEQ ID NOs: 19-36.
  • the digital PCR or ddPCR may be performed with probes comprising each of SEQ ID NOs: 19-36.
  • the instant methods comprise determining the presence of a mutation in histone H3 (e.g., histone H3.3).
  • the mutation in histone H3 is K27M.
  • the histone H3 is H3F3A.
  • the method comprises determining the presence of a mutation in H3F3A and at least one other gene (e.g., partner gene).
  • the other gene(s) may be, for example, selected from the group of HIST1H3B (e.g., p.K27M), HIST2H3C (e.g., p.K27M), ACVR1 (e.g., p.G328V/p.R206H), PPM1D (e.g., p.E525X), PIK3R1 (e.g., p.K567E), PIK3CA (e.g., p.H1047R), IDH1 (e.g., p.R132H; isocitrate dehydrogenase (NADP(+)) 1, cytosolic; Gene ID: 3417) and BRAF (e.g., p.V600E; B-Raf proto-oncogene, serine/threonine kinase
  • the other gene(s) is selected from the group of HIST1H3B (e.g., p.K27M), ACVR1 (e.g.,
  • p.G328V/p.R206H PPM1D (e.g., p.E525X), and PIK3R1 (e.g., p.K567E) (e.g., 1, 2, 3, or all 4 genes are analyzed). Sequence information for certain of these genes is provided below.
  • H3 histone family member 3A (H3F3A; SEQ ID NO: 37) Gene ID: 3020, Gen Bank Accession Nos. NM_002107.4 and NP_002098.1 (Note: the Met at position 1 is not included in amino acid numbering)
  • H3 family member b HIST1H3B; SEQ ID NO: 38
  • ACVRl Activin A receptor type 1 (ACVRl ; SEQ ID NO: 39) Gene ID: 90, Gen Bank Accession
  • Protein phosphatase, Mg2+/Mn2+ dependent ID (PPM1D; SEQ ID NO: 40) Gene ID: 84, Gen Bank Accession Nos. NM bend003620.3 and NPJ303611.1
  • Phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1 ; SEQ ID NO: 41) Gene ID: 529 Gen Bank Accession Nos. NM_181523.2 and NP_852664.1
  • Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA; SEQ ID NO: 42) Gene ID: 5290, Gen Bank Accession Nos. NM_006218.3 and NP_006209.2
  • the method comprises 1) administering a cancer therapy to a subject in need thereof, 2) obtaining a biological sample from the subject after administration of the cancer therapy to the subject, and 3) determining the presence of a mutation in in the biological sample (e.g., in histone H3 (e.g., histone H3.3)).
  • the method may comprise obtaining multiple biological samples over time from the subject after administration of the cancer therapy to the subject.
  • the method may comprise obtaining a biological sample from the subject before administration of the cancer therapy to the subject (e.g., a baseline).
  • the mutation e.g., in histone H3
  • the cancer therapy administered to the subject is effective (e.g., inhibiting tumor growth).
  • the biological sample of the methods may be a fluid or liquid such as blood, CSF, plasma, serum, or urine.
  • the biological sample is blood or plasma.
  • the cancer monitored by the instant methods may be a brain tumor. More specifically, the cancer may be a pediatric brain tumor. In a particular embodiment the cancer is a glioma, particularly a diffuse glioma or a diffuse intrinsic pontine glioma (DIPG).
  • DIPG diffuse intrinsic pontine glioma
  • the cancer therapy administered to the subject may comprise any type of therapy.
  • the cancer therapy may comprise the administration of at least one chemotherapeutie agent (e.g., a small molecule).
  • the cancer therapy may include radiation therapy.
  • the methods of the instant invention may comprise determining the presence of the mutation in circulating tumor DNA (ctDNA).
  • determining of the presence of the mutation comprises performing digital PGR.
  • the determining of the presence of the mutation comprises performing droplet digital PGR (ddPCR).
  • the digital PGR or ddPCR may be performed with at least one primer comprising any one of SEQ ID NOs: 1-14 or a portion thereof.
  • the digital PGR or ddPCR may be performed with at least one probe comprising any one of SEQ ID NOs: 19-36.
  • the digital PGR or ddPCR may be performed with probes comprising each of SEQ ID NOs: 19-36.
  • the instant methods comprise determining the presence of a mutation (e.g., in histone H3, such as histone H3.3).
  • the method comprises determining the presence of a mutation in a gene(s) selected from the group of H3F3 A (e.g., p.K27M), HIST1H3B (e.g., p.K27M), HIST2H3C (e.g., p.K27M), ACVR1 (e.g., p.G328V/p.R206H), PPM1 (e.g., p.E525X), FK3R1 (e.g., p.
  • H3F3 A e.g., p.K27M
  • HIST1H3B e.g., p.K27M
  • HIST2H3C e.g., p.K27M
  • ACVR1 e.g., p.G328V/p.R206H
  • PPM1 e.
  • PIK3CA e.g., p.H1047R
  • IDH1 e.g., p.R132H; isocitrate dehydrogenase (NADP(+)) 1, cytosolic (Gene ID:3417)
  • BRAF e.g., p.V600E; B-Raf proto-oncogene, serine/threonine kinase (Gene ID: 673)
  • the gene(s) is selected from the group of H3F3A (e.g., p.K27M), HIST1H3B (e.g., p.K27M), ACVR1 (e.g.,
  • the other gene(s) is selected from the group of HIST1H3B (e.g., p.K27M), ACVR1 (e.g.,
  • the mutation in histone H3 is K27M.
  • the histone H3 is H3F3A.
  • the method comprises determining the presence of a mutation in H3F3A and at least one other gene. A decrease in the presence of the mutations over time (e.g., when multiple biological samples are obtained after therapy) or compared to the amount observed prior to therapy, indicates that the cancer therapy administered to the subject is effective (e.g., inhibiting tumor growth).
  • oligonucleotide probes are also provided with the instant invention.
  • the probes are designed to have high affinity and specificity to the target site (e.g., the mutations set forth herein and, optionally, the wild- type gene).
  • oligonucleotide probe(s) target a gene (e.g., wild-type and/or mutant) selected from the group of H3F3A (e.g., p.K27M), HIST1H3B (e.g., p.K27M), HIST2H3C (e.g., p.K27M), ACVR1 (e.g., p.G328V/p.R206H), PPM1D (e.g., p.E525X), PIK3R1 (e.g., p.K567E), PIK3CA (e.g., p.H1047R), IDH1 (e.g., p.R132H) and BRAF (e
  • the probes do not have an absolute requirement on length. However, the probes will typically be from about 10 to about 250 nucleotides, about 10 to about 100, about 10 about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 25 nucleotides, or about 10 to about 20 nucleotides. In a particular embodiment, the probe is at least about 10 nucleotides in length. The probe may be at least 85%, at least 90%, at least 95%, at least 97%, or, more preferably, 100% complementary to the target sequence. In a particular embodiment, the oligonucleotide probe is designed such that the mutation is towards the middle of the sequence of the probe (e.g., within the middle third of the probe length).
  • the probe may comprise at least one nucleotide analog.
  • the nucleotide analogs may be used to increase annealing affinity and/or specificity and/or resistance to degradation.
  • LNA locked nucleic acid
  • Nucleotide analogs include, without limitation, nucleotides with phosphate modifications comprising one or more phosphorothioate, phosphorodithioate, phosphodiester, methyl phosphonate, phosphoramidate, methylphosphonate, phosphotriester, phosphoroaridate, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl substitutions; nucleotides with modified sugars; and nucleotide mimetics such as, without limitation, peptide nucleic acids (PNA), morpholino nucleic acids, cyclohexenyl nucleic acids, anhydrohexitol nucleic acids, glycol nucleic acid, threose nucleic acid, and locked nucleic acids (LNA).
  • PNA peptide nucle
  • the probes comprise at least one locked nucleic acid.
  • the probes may comprise one of SEQ ID NOs: 19-36 or a sequence with at least 85%, at least 90%, at least 95%, or at least 97% identity to one of SEQ ID NOs: 19-36.
  • the probe comprises one of SEQ ID NOs: 19-36.
  • the probes may comprise additional nucleotides 5' or 3' to the included SEQ ID NO.
  • the probe may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides 5' or 3' to the included SEQ ID NO.
  • the additional sequences are complementary to the target sequence.
  • the probes of the instant invention may comprise one or more fluorescent probes / fluorophores and/or quenchers.
  • the fluorophores and or quenchers may be added to the 5' and or 3' termini of the probes.
  • the fluorophores and/or quenchers may also be added to internal part of the probes (e.g., a ZEN probe). Fluorophores and/or quenchers are well known in the art (see, e.g., IDT, Coralville, IA).
  • fluorophores and/or quenchers include, without limitation, 6-FAM, fluorescein, Cy3, Cy5, TAMRA, JOE, MAX, TET, Cy5.5, ROX, ATTO, TYE, Yakima Yellow®, HEX, TEX, Texas Red®, Iowa Black®, ZEN, and Alexa Fluor®.
  • the fluorophores and/or quenchers used allow for the determination of the presence of the wild- type and/or mutant allele in a sample at the same time (see, e.g., the Examples).
  • the fluorophores and/or quenchers create an energy transfer pair (e.g., fluorescence resonance energy transfer (FRET)) (e.g.,as set forth in Table 1).
  • FRET fluorescence resonance energy transfer
  • the probes comprise a fluorophore and/or quencher combination presented in Table 1.
  • the probe comprises any one of SEQ ID NOs: 19-36.
  • the probe comprises any one of SEQ ID NOs: 19-36 along with the modifications presented in Table 1.
  • compositions comprising at least one probe of the instant invention are also provided.
  • the composition is an aqueous solution.
  • the composition comprises at least one probe comprising any one of SEQ ID NOs: 19-36.
  • the composition comprises individual probes comprising each of SEQ ID NOs: 19-36.
  • compositions comprising at least one primer of the instant invention are also provided.
  • the composition is provided an aqueous solution.
  • the composition comprises at least one primer comprising any one of SEQ ID NOs: 1-18.
  • the composition comprises individual primers comprising each of SEQ ID NOs: 1-18.
  • “Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • a “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g.,Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris HC1, acetate, phosphate), antimicrobial, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle (e.g., with which an active agent of the present invention is administered).
  • Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin.
  • Water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions.
  • Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of
  • small molecule refers to a substance or compound that has a relatively low molecular weight (e.g., less than 4,000, less than 2,000, particularly less than 1 kDa or 800 Da).
  • small molecules are organic, but are not proteins, polypeptides, or nucleic acids, though they may be amino acids or dipeptides.
  • treat refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.
  • prevent refers to the prophylactic treatment of a subject who is at risk of developing a condition resulting in a decrease in the probability that the subject will develop the condition.
  • diagnosis refers to detecting and identifying a disease or disorder in a subject.
  • the term may also encompass assessing or evaluating the disease or disorder status (progression, regression, stabilization, response to treatment, etc.) in a patient known to have the disease or disorder.
  • the term "prognosis” refers to providing information regarding the impact of the presence of a disease or disorder (e.g., as determined by the diagnostic methods of the present invention) on a subject's future health (e.g., expected morbidity or mortality, the likelihood of getting or risk of cholestasis).
  • a disease or disorder e.g., as determined by the diagnostic methods of the present invention
  • future health e.g., expected morbidity or mortality, the likelihood of getting or risk of cholestasis.
  • prognosis refers to providing a prediction of the probable course and outcome of a disease/disorder and/or the likelihood of recovery from the disease/disorder.
  • the term "subject" refers to an animal, particularly a mammal, particularly a human.
  • composition refers to an amount effective to prevent, inhibit, treat, or lessen the
  • the treatment of a disease or disorder herein may refer to curing, relieving, and/or preventing the disease or disorder, the symptom(s) of it, or the predisposition towards it.
  • therapeutic agent refers to a chemical compound or biological molecule including, without limitation, nucleic acids, peptides, proteins, and antibodies that can be u ed to treat a condition, disease, or disorder or reduce the symptoms of the condition, disease, or disorder.
  • isolated refers to the separation of a compound from other components present during its production or from its natural environment. "Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not substantially interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, or the addition of stabilizers.
  • a “biological sample” refers to a sample of biological material obtained from a subject, particularly a human subject, including a tissue, a tissue sample, cell(s), and a biological fluid (e.g., blood (e.g., whole blood), serum, plasma, urine, sweat, tears, saliva, mucosal secretions, sputum, CSF).
  • a biological fluid e.g., blood (e.g., whole blood), serum, plasma, urine, sweat, tears, saliva, mucosal secretions, sputum, CSF).
  • probe refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe.
  • a probe may be either single- stranded or double- stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method.
  • the oligonucleotide probe typically contains about 10-250, about 10-100, about 10-50, about 15-30, about 15-25, or about 10-20 nucleotides.
  • the probes herein may be selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically
  • the probe sequence need not reflect the exact complementary sequence of the target, although they may.
  • a non-complementary nucleotide fragment may be attached to the 5' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand.
  • non- complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.
  • a probe may be tagged or labeled (i.e., attached to an entity making it possible to identify a compound to which it is associated (e.g., fluorescent or radioactive tag).
  • a label is selected from the group consisting of biotin, copper-DOTA, biotin-PEG3, aminooxyacetate, 19 FB, 18 FB, FITC-PEG 3 , fluorescein and fluorescein derivatives (e.g., 5-carboxy fluorescein).
  • the label is selected from the group consisting of 64 Cu DOT A, 68 Ga DOT A, 18 F, ⁇ C , 68 Ga, 89 Zr, i24 I, 86 Y, 94m Tc, n0ra Xn, "C and 76 Br.
  • primer refers to an oligonucleotide, either RN A or DNA, either single- stranded or double-stranded, which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis.
  • suitable nucleoside triphosphate precursors of nucleic acids a polymerase enzyme, suitable cofactors and conditions such as appropriate temperature and H
  • the primer may be extended at its 3' terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product.
  • the primer may vary in length depending on the particular conditions and
  • the oligonucleotide primer is typically about 10- 25 or more nucleotides in length, but can be significantly longer.
  • the primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able to anneal with the desired template strand in a manner sufficient to provide the 3' hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template, though it may.
  • a non- complementary nucleotide sequence may be attached to the 5' end of an otherwise complementary primer.
  • non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.
  • oligonucleotide refers to sequences, primers and probes of the present invention, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.
  • the respective fluorophores are added to the 5' end of the probes, and the quenchers are added to the middle and at the 3' end of the probe.
  • the symbol + following a nucleotide indicates a locked nucleic acid.
  • IABkFQ Iowa Black® FQ.
  • SEQ ID NO is provided in parentheses. The SEQ ID NOs represent the nucleotide sequences without the probes or modifications.
  • FIG. 1A This example illustrates a sensitive and specific platform for discriminating rare, low abundant, tumor-associated circulating DNA in pediatric patients with mid-line glioma (MLG) tumors.
  • MLG mid-line glioma
  • Fig. 1A Clinicopathological and genomic characteristics of the tumors of a pediatric MLG patient cohort are shown in Fig. 1A.
  • Patients were diagnosed with MLGs harboring various mutation combinations, including oncohistone variants: 94% harbored histone 3 mutations (79% with H3.3K27M, 15% with H3.1K27M), and 6% were H3 wild type.
  • the initial goal was to develop and validate a clinically relevant and minimally invasive Uquid biopsy platform suitable for detection and quantification of somatic mutations associated with pediatric MLGs.
  • Example 2 CSF and plasma harbor circulating tumor DNA indicative of driver mutations associated with pediatric MLGs.
  • Liquid biome specimens were analyzed from 84 subjects (48 MLG patients, and 36 non- CNS diseased controls), enrolled in an ongoing clinical trial PNOC003 (NCT 227498), and consented for the Children's National (CN) brain tumor biorepository (FIG. IB).
  • CSF samples were collected at a single time point through the CN biorepository at pre-treatment, during therapy, and at postmortem from 27 MLG patients, while serial sampling at pre-treatment and postmortem was available for one patient with DIPG.
  • Histone 3 mutant and wild type alleles were detected in 75% of CSF specimens collected at diagnosis, 67% of those collected during treatment, and 90% of those collected at postmortem (Fig. 2a).
  • H3 K27M- mutant ctDNA was detected in 89% of all CSF specimens analyzed from 27 MLGs, where 23 of these were confirmed to harbor oncohistones as assessed by tumor DNA analysis.
  • Example 3 Assessment of treatment response using ctDNA quantification.
  • Liquid biopsy is an emerging tool for diagnosing, and measuring efficacy of treatment in adult cancer patients. While molecular profiling of tumors is a localized method, a liquid biopsy approach provides a systemic molecular overview. ctDNA has been used to determine patient's mutation profiles, as a biomarker for molecular-based disease monitoring, and recurrence in adult chronic lymphocytic leukemia, breast, and colon cancer. 13 ' 16 ' 23 The only previously reported liquid biopsy approach for pediatric MLGs was established using Sanger sequencing.
  • the disclosure herein for the detection and quantification of tumor-associated circulating DNA using ddPCR allows for rapid, more sensitive, far less costly and less invasive method for surveying tumor mutations, and represents a key advance particularly for tumors with limited tissue acquisition or prohibitive sampling at multiple time points.
  • DIPG tumor cells disseminate throughout the brain during the course of disease.
  • Our ctDNA analysis in DIPG patients was indicative of tumor expansion beyond pons, where an increased amount of ctDNA in CSF was observed in patients who exhibited tumor spread.
  • Studies of a larger cohort in clinical settings are required to assess the statistical significance of our finding.
  • our results indicate the unique strength of liquid biopsy for assessing the molecular landscape of MLGs, and potential for longitudinal assessment of tumor response to therapy, which is a new tool that is complementary to MR imaging. Similar to the clinical utility of ctDNA for monitoring response to therapy with respect to MRIs in adult GBMs, despite a small sample size, we found significant reduction in ctDNA following RT.
  • Table 2 Assessing specificity of a ddPCR platform by testing non-CNS malignant pediatric CSF and plasma.
  • Average MAF values for non- CNS diseased plasma analyzed for H3F3A p.K27M mutation represent technical triplicates, all other average MAF values for plasma and CSF analyzed for H3F3A and HIST1H3B p.K27M represent technical duplicates.

Abstract

L'invention concerne des biomarqueurs tumoraux circulants, ainsi que des procédés d'utilisation de ceux-ci.
PCT/US2018/051150 2017-09-15 2018-09-14 Procédés de détection de biomarqueurs du cancer WO2019055829A1 (fr)

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