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WO2018183796A1 - Systèmes et procédés de prédiction et de surveillance d'une thérapie anticancéreuse - Google Patents

Systèmes et procédés de prédiction et de surveillance d'une thérapie anticancéreuse Download PDF

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WO2018183796A1
WO2018183796A1 PCT/US2018/025335 US2018025335W WO2018183796A1 WO 2018183796 A1 WO2018183796 A1 WO 2018183796A1 US 2018025335 W US2018025335 W US 2018025335W WO 2018183796 A1 WO2018183796 A1 WO 2018183796A1
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fluid
methylation
rna
cdna
dsdna
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Zhixin Zhao
Shidong JIA
Pan DU
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Predicine Inc
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Predicine Inc
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Priority claimed from PCT/US2017/027830 external-priority patent/WO2017181161A1/fr
Priority claimed from PCT/US2017/052832 external-priority patent/WO2018057820A1/fr
Application filed by Predicine Inc filed Critical Predicine Inc
Priority to US16/499,449 priority Critical patent/US20210102256A1/en
Publication of WO2018183796A1 publication Critical patent/WO2018183796A1/fr
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    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates generally to the field of precision medicine, specifically cancer prediction, diagnostics or prognostics, and more specifically of methods for predicting cancer development in cancer patients by the detection of methylation and gene expression of essential genes for cancer treatment using human biofluid samples, e.g., plasma, urine, CSF and saliva.
  • human biofluid samples e.g., plasma, urine, CSF and saliva.
  • Cancer immunotherapy is a treatment that involves components of the immune system in human bodies. Most immunotherapies use antibodies which bind to the proteins expressed by cancer cells, therefore, inhibiting the protein function.
  • FDA has approved inhibitors of either the programmed death receptor (PD-1) or its ligand (PD-Ll) for treatment of cancer patients with metastatic melanoma, metastatic non-small cell lung cancer, recurrent or metastatic head and neck cancer and urothelial carcinoma.
  • PD-1 programmed death receptor
  • PD-Ll its ligand
  • FDA approved the pembrolizumab (anti- PD-1 antibody) for any unresectable or metastatic solid tumor with microsatellite instability (MSI) or mismatch repair deficiency (MMR).
  • MSI microsatellite instability
  • MMR mismatch repair deficiency
  • PD-1(CD279) is primarily expressed on the surface of activated T-cells and PD-Ll (CD274) is the main ligand of PD-1.
  • PD-1/PD-L1 binding inhibits the function of activated T- cells.
  • High level of PD-Ll from the cancer cells helps cancer cells evade immune attack by binding PD-1 and inhibiting of T-cell activation.
  • One important clinical question regarding the PD-l/PD- Ll directed immunotherapy is the uncertainty of the clinical outcome.
  • the most popular biomarker for anti-PD-l/PD-Ll therapy is PD-Ll detection by immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • Companion diagnostic PD-Ll IHC tests for anti-PD-1 therapy have been approved by FDA (PD- LI IHC 22C3 pharmDx, DAKO/AGILENT TECHNOLOGIES).
  • next generation sequencing is accelerating the discovery of genetic and epigenetic alteration in human diseases.
  • NGS next generation sequencing
  • One of the major advantages of next generation sequencing is multiplex sequencing. NGS technology also enables adding a molecular barcode to identify the source of the NGS reads.
  • the present invention provides a method for diagnosing a disease status or progression of a disease comprising 1) assaying the methylation and expression status of one or more cancer markers in a biofluid sample from a subject, and 2) detecting the presence of one or more methylation sites of the cancer marker and level of RNA expression of the cancer marker; wherein the correlation of the methylation level and RNA expression changes indicates the disease status.
  • the cancer markers include but are not limited to PD-L1 or other cancer markers known in the art.
  • the disease status including the presence or absence of the disease, the progress of the disease, the prediction of the disease.
  • the present invention provides an accurate diagnosis than other known tools.
  • the method employs an approach to isolate RNA from circulating tumor RNA (ctRNA) and RNA-containing extracellular vesicles in a biofluid sample.
  • the biofluid sample can be a sample of blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid, or a combination thereof.
  • the disclosed method comprises assaying the methylation and expression status of PD-L1 gene in a biofluid sample from the patient; wherein the presence of one or more CG methylation sites and low PD-L1 expression indicates the reduced immunotherapy output.
  • the means for assaying the methylation and expression status of PD-L1 gene comprises a) extracting cell-free nucleic acid from a biofluid sample of the patient, b) bisulfite treatment of DNA, c) barcoding the DNA and RNA, and d) bioinformatics assaying of the barcoded DNA and RNA related to PD-L1 gene.
  • the method is for predicting immunotherapy in cancer patients.
  • the present invention disclosed a method of combined DNA and RNA assays comprising a step of bioinformatics analysis identifying genetic and epigenetic biomarker signatures that enables prospective prediction of therapies in a cancer patient.
  • the method comprises assaying the presence or absence of CpG methylation of cancer markers in a biofluid sample from the patient; wherein the presence of the one or more CpG methylation site and low PD-L1 RNA expression level indicate the presence of poor clinical outcome of the anti-PD-l/PD- LI therapy in cancer patients.
  • kits for detecting methylation and expression of cancer markers in a patient comprising: (a) reagents for circulating nucleic acid extraction; (b) reagents for bisulfite conversion; and (c) reagents for reverse transcription; (d) reagents for library preparation; (e) reagents for MSP and/or ddPCR; (f) and/or oligos targeting cancer marker genes.
  • the kit is used for detecting CpG methylation and expression of cancer marker genes by NGS.
  • the kit is used for detecting CpG methylation and expression of PD- Ll by NGS.
  • the kit is used for detecting specific CpG methylation site and gene expression of cancer markers by ddPCR.
  • the disclosed method comprises assaying the presence or absence of one or more epigenetic alteration such as DNA methylation and presence or absence of one or more genetic alterations such as gene splice variants, mutation, indels, long deletions, copy number variation, gene fusions etc. in a biofluid sample from the said patient.
  • epigenetic alteration such as DNA methylation
  • genetic alterations such as gene splice variants, mutation, indels, long deletions, copy number variation, gene fusions etc.
  • the present invention further discloses Gene RADAR (RNA and DNA single molecular digital Reading) assay and bioinformatics analysis tool for combined genetics and epigenetics alteration analysis.
  • the bioinformatics analysis enables 1) differentiation of the RNA derived reads from DNA derived reads by checking the RNA specific tags in the sequence reads; 2) the suppression of the sequencing and background noise by creating consensus of Next Generation Sequence (NGS) reads from the same original molecules, which is defined based on molecular barcodes and the mapping location of the reads; and 3) accurate quantification of RNA by combining two types of barcodes (RNA specific molecular barcodes + DNA molecular barcodes), and the quantification of DNA (only using DNA molecular barcodes) at the same time; and 4) detection of epigenetic alteration by quantification of DNA methylation.
  • NGS Next Generation Sequence
  • FIG. 1 shows an assay flowchart depicting the steps from plasma nucleic acid extraction to NGS and ddPCR assay of methylation and gene expression.
  • FIG. 2A-2D show the negative correlation between PD-Ll methylation and gene expression.
  • FIG. 2A shows PD-Ll DNA methylation and mRNA expression level are negatively correlated based on TCGA data.
  • FIG. 2B we performed ddPCR assay for PD-Ll gene expression (FIG. 2B) and methylation NGS assay for methylation status of PD-Ll (FIG. 2C) and the results showed the methylation and expression of PD-Ll in these two samples are negatively correlated (FIG. 2D).
  • the present invention provides a method for diagnosing a disease status or progression of a disease comprising 1) assaying the methylation and expression status of one or more cancer markers in a biofluid sample from a subject, and 2) detecting the presence of one or more methylation sites of the cancer marker gene and level of RNA expression of the cancer marker; wherein the correlation of the methylation level and RNA expression changes indicates the disease status.
  • the cancer marker genes include but are not limited to DNA, variations, genetic alterations, modifications and any changes made but remain the main characteristics of the gene itself.
  • the cancer markers include but are not limited to PD-L1 or other cancer markers known in the art.
  • the disease status including the presence or absence of the disease, the progress of the disease, the prediction of the disease.
  • the present invention can also be combined with other known tools to aid in the determination of the disease status.
  • the disease includes but not limiting to tumor, cancer or other diseases that related to the cancer makers.
  • the disease status includes but not limiting to the stage of the disease, the progress of the disease.
  • the diagnosing a disease includes but not limiting to the detecting a disease, detecting a disease status by using companion diagnostics method, detecting the disease status by using a condition including but not limiting to drug treatment or using other means.
  • the present invention discloses a novel method for identifying genetic and epigenetic biomarker signatures that enables prospective prediction of resistance to anti-PD-l/PD-Ll therapies in a cancer patient.
  • the present invention also disclosed a gene panel of multiple mixed genes for predicting and monitoring cancer immunotherapy.
  • FIG. 1 shows the flowchart including the steps from plasma nucleic acid extraction to NGS and/or ddPCR assay.
  • the present disclosure allows the detection of methylation and expression of interesting genes from biofluids in specimens of various sources.
  • biofluids There are abundant ctRNA and extracellular vesicles in cancer patients and the abundance of ctRNA and extracellular vesicles are independent of the number of CTCs.
  • the present method enables the detection of genetic alterations in more patients than CTC-based methods.
  • the disclosed method streamlines the extraction of circulating DNA and RNA from biofluids, bisulfite conversion of the DNA, reverse transcription of the RNA and NGS library preparation and sequencing. In contrast to existing methods known in the art, this streamlined process enables the non-invasive detection of methylation and gene expression of interest by using biofluid samples in a quick, simplified, and consistent manner.
  • the present disclosure provides a method for detecting various solid cancers in a patient comprising assaying the presence or absence of CpG methylation site of PD- Ll and expression of interesting immunotherapy genes in a biofluid sample from the patient.
  • the step of assaying comprises extracting RNA from the biofluid sample and subsequently reverse transcribing the extracted RNA into a complementary DNA (cDNA).
  • the step of assaying comprises extracting both DNA and RNA from the biofluid sample simultaneously and then bisulfate convert the extracted DNA into single strand DNA (ssDNA).
  • the resultant ssDNA is subsequently used to detect the PD-L1 methylation by Methylation-Specific Polymerase Chain Reaction (MSP).
  • MSP Methylation-Specific Polymerase Chain Reaction
  • the resultant ssDNA is subsequently measured by Next Generation Sequencing and/or Polymerase Chain Reaction.
  • the resultant cDNA is subsequently used to detect the PD-L1 expression by digital droplet Polymerase Chain Reaction (ddPCR).
  • ddPCR digital droplet Polymerase Chain Reaction
  • the resultant cDNA is subsequently measured by Next Generation Sequencing. In some embodiments, the resultant cDNA is subsequently measured by Polymerase Chain Reaction.
  • the system can be a closed system and an automated system.
  • the terms “comprises,” “comprising,” “includes,” “including,” “contains,” “containing,” and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, product-by-process, or composition of matter that comprises, includes, or contains an element or list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, product-by-process, or composition of matter.
  • patient preferably refers to a human, but also encompasses other mammals. It is noted that, as used herein, the terms “organism,” “individual,” “subject,” or “patient” are used as synonyms and interchangeably.
  • genetic alteration comprise gene splice variants, SNV, Indel, CNV, fusion and combination thereof.
  • gene expression means transcription and/or translation of genes. Specifically herein, gene expression is the transcription of DNA to RNA.
  • circulating tumor DNA circulating tumor RNA
  • ctDNA or circulating tumor RNA is tumor- derived fragmented DNA or RNA in the bloodstream that is not associated with cells.
  • ctDNA or ctRNA should not be confused with cell-free DNA (cfDNA) or cell-free RNA (cfRNA), a broader term which describes DNA or RNA that is freely circulating in the bloodstream, but is not necessarily of tumor origin.
  • cell-free DNA cfDNA
  • cell-free RNA cfRNA
  • cfDNA DNA or RNA freely circulating in the bloodstream and not associated with cells.
  • barcoding means using one or more oligonucleotides as tags/markers to incorporate into a dsDNA.
  • the barcodes will be sequenced together with the unknown sample DNA. After sequencing the reads are sorted by barcode and grouped together (de-multiplexing). Barcode includes molecular barcode and sample barcode.
  • a "molecular barcode” is a unique multiple-base pair sequence used to identify unique fragments and “de-duplicate” the sequencing reads from a sample. This, along with the random start sites, helps identify and remove PCR duplicates. Molecular barcodes can be used to suppress sequencing and PCR errors, and reduce false positives subsequently. Whereas sample barcodes, also called indexed adaptors, are customarily used in most current NGS workflows and allow the mixing of samples prior to sequencing.
  • RNA molecular barcoding means incorporating barcodes during the process of reverse transcription of RNA and ds-cDNA library preparation.
  • RNA molecular barcoding can incorporate a molecular barcode or multiple molecular barcodes.
  • An RNA specific barcode can be an RNA specific tag, a molecular barcode, a sample barcode or a combination.
  • DNA barcoding means barcoding at dsDNA level with a multiple-base pair sequence that is part of the adapter for multiplex sequencing.
  • the adapter is designed in-house.
  • the incorporated DNA barcodes can be molecular barcodes alone or both molecular barcodes and sample barcodes.
  • positive strand also known as the "sense” strand or coding strand
  • negative strand also known as the "anti-sense” strand of DNA is the segment within double-stranded DNA that runs from 3' to 5'.
  • bioinformatics means a sequencing analysis tool/software including but are not limited to Gene RADAR software or any software that can analyze DNA/RNA sequencing results.
  • Cancer markers include but are not limited to PD1, PD-L1 and PD-L2, human stratum corneum chymotryptic enzyme (HSCCE), kallikrein 4, kallikrein 5, kallikrein 6 (protease M), kallikrein 8, kallikrein 9, kallikrein 10, CA125, CA15-3, CA19-9, OVX1, lysophosphatidic acid(LPA), carcinoebryonic antigen (CEA), macrophage colonystimulating factor (M-CSF), prostasin, CA54-61, CA72, HMFG2, IL-6, IL-10, LSA, M-CSF, NB70K, PLAP, TAG72, TNF, TPA, UGTF, WAP four-disulfide core domain 2 (HE4), matrix metalloprotease 2, tetranectin, inhibin, mesothelyn, MUC1, VEGF, CLDN3, NOTCH3, E2F transcription
  • HSCCE
  • the present invention provides a method for barcoding an oligonucleotide tag on the RNA sample during reserve transcribing it to cDNA and ds-cDNA, wherein the reverse transcription step of the ssRNA includes 1) reverse transcribing ssRNA to cDNA using a gene-specific or random primer annealed to an oligonucleotide comprising a RNA specific tag and random molecular barcodes; and/or 2) converting the cDNA to a ds-cDNA.
  • converting the cDNA to a ds-cDNA is conducted by annealing a non-coded primer, wherein such barcoding step is named single-sided RNA barcoding.
  • the RNA specific tag comprising an oligonucleotide.
  • the random molecular barcodes comprise another oligonucleotide.
  • the oligonucleotide consists of 5, 8, 10, 12, 14, 15, 20 nucleic acid bases.
  • the oligonucleotide can be designed for fitting the identification in further analysis.
  • the converting step of the cDNA to a ds-cDNA is conducted by annealing an oligonucleotide comprising a second RNA specific molecular barcode, wherein such barcoding step is named double-sided RNA barcoding.
  • the first and second RNA specific molecular barcode is the same; in some other embodiment, the first RNA and second RNA specific molecular barcode is not the same.
  • the genetic alterations include gene splice variants, mutations, indels, long deletions, copy number changes, fusions and combination thereof.
  • the method of detecting the alterations is used to detect the changes of above.
  • the barcoded signals from RNA and DNA are read by next generation sequencing. Separation of RNA and DNA derived reads is conducted with the Gene RADAR bioinformatics analysis tool.
  • RNA molecular barcodes and DNA molecular barcodes will be utilized to recognize the reads from RNA or DNA with barcode matches. Then the recognized DNA reads are mapped to the genome, while the recognized RNA reads are mapped to transcriptome and genome. Barcode consensus is created by merging NGS reads originally from the same molecule (identified based on molecular barcodes and genome mapping location of the reads). The sequencing and PCR errors can be corrected or marked when there are inconsistent variants originally from the same molecule.
  • the genetic alteration of DNA includes SNV, Indel, long deletion, CNV and DNA fusion.
  • the genetic alteration of RNA includes splicing, fusion, SNV, Indel analysis.
  • the epigenetic alteration of DNA includes methylation of one or more CpG sites. Then the DNA and RNA analysis results are integrated to achieve comprehensive reporting of genetic and epigenetic alterations.
  • barcode or barcoding with random oligonucleotide sequences such as 5, 8, 10 12, 14, 15 nucleotides to uniquely tag individual target DNA molecules can be used.
  • the oligonucleotide can be designed for fitting the identification in further analysis. Such application increases the sensitivity and reduces false positives. For example, it can be used for PCR or NGS analysis to identify individual molecules (DNA or RNA fragments) in samples.
  • the Gene RADAR detects DNA methylation at CpG sites and DNA level variants while measuring other RNA expression and RNA level variants including splicing, fusion, SNV, Indel at the same time from the patient biofluid sample.
  • the present invention further provides a platform for detecting multiple gene variants in a patient, including: (a) a kit of reagents for circulating nucleic acid extraction; (b) barcoding sequences for two-layer RNA+DNA molecular barcoding; and (c) bioinformatics tool to analyzing DNA and RNA-derived information.
  • the system can be an opened or closed system. And both systems can be an automated system.
  • the system can be in a device setting.
  • the detection of presence or absence of a genetic alteration and epigenetic alteration is indicative of the immunotherapy resistance to a disease and the disease is one or more cancers.
  • presence or absence of multiple genetic alteration and presence/absence of one or more epigenetic alteration is indicative of a disease and the disease is one or more cancers.
  • presence/absence of multiple epigenetic alteration and presence/absence of one or more genetic alteration are indicative of a disease and the disease is one or more cancers.
  • the samples include but are not limited to blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid, and a combination thereof.
  • the detected genetic alteration and epigenetic alteration information can be used to detect immunotherapy-resistant cancer in a patient comprising assaying the presence or absence of one or more types of genetic alterations and epigenetic alterations at both RNA and DNA levels, such as PD-L1 gene DNA methylation and RNA/DNA-based mutation detection in a biofluid sample from the patient; wherein the presence of such genetic and epigenetic alterations indicates the presence of immunotherapy-resistant cancer in the patient.
  • RNA is extracted together with DNAs from circulating nucleic acid and nucleic acid-containing extracellular vesicles in a biofluid sample.
  • the sources of nucleic acids are extracellular vesicles (EVs), including exosomes and microvesicles, which have been shown to carry a variety of biomacromolecules including mRNA, microRNA and other non-coding RNAs and considered to be a minimally invasive novel source of materials for molecular diagnostics. See Jia et al., "Emerging technologies in extracellular vesicle-based molecular diagnostics", Expert Rev. Mol. Diagn. 1-15 (2014).
  • EVs are membranous, cell-derived, mixed populations of vesicles, ranging from approximately 40-5000 nm in diameter, which are released by a variety of cells into the intercellular microenvironment and various extracellular biofluids.
  • Methods for procuring a microvesicle fraction from a biofluid sample are described in scientific publications and patent applications (Chen et al., 2010; Miranda et al., 2010; Skog et al., 2008). See also WO 2009/100029, WO 2011009104, WO 2011031892, and WO 2011031877.
  • methods of microvesicle procurement by differential centrifugation are described in a paper by Raposo et al.
  • microvesicles can be identified and isolated from a subject's bodily fluid by a microchip technology that uses a microfluidic platform to separate tumor-derived microvesicles (Chen et al., 2010).
  • Methods for nucleic acid extraction are generally based on procedures well-known in the art plus proprietary procedures developed in-house. Persons of skill will select a particular extraction procedure as appropriate for the particular biological sample. Examples of extraction procedures are provided in patent publications WO/2009/100029, US 20100196426, US 20110003704, US 20110053157, WO 2011009104, WO 2011031892, US20130131194 and US20110151460. Each of the foregoing references is incorporated by reference herein for its teaching of these methods.
  • biofluids contain circulating nucleic acids and/or nucleic acid-containing EVs.
  • these biofluids include blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid, or a combination thereof.
  • the biofluid sample is obtained from a subject who has been diagnosed with cancer according to tissue or liquid biopsy and/or surgery or clinical grounds.
  • Example 1 Extracting cell-free Nucleic Acid (cfNA) from human bio-fluid samples, e.g., plasma, urine and saliva and using Gene RADAR technology to split the extracted the cfNA as cfDNA and cfRNA.
  • cfNA cell-free Nucleic Acid
  • cfDNA Single-strand DNA(ssDNA) by DNA bisulfite treatment.
  • the ssDNA can be used as a template for methylation-specific PCR (MSP) of PD-Ll; the ssDNA can also be used for an ssDNA library preparation and a small portion of the ssDNA library can be used for PD-Ll MSP.
  • MSP methylation-specific PCR
  • the ssDNA library can be enriched using Methylation panel, which contains the CpG methylation sites of PD-Ll gene.
  • cfRNA was reverse transcribed into single-strand cDNA. So the cDNA can be used as a template for PD-Ll ddPCR; the cDNA can also be used for cDNA library preparation and a small portion of the cDNA library can be used for PD-Ll ddPCR.
  • the cDNA library can be enriched using Cancer immunotherapy panel of mixed genes.
  • Enriched ssDNA and cDNA libraries are barcoded with different barcodes and loaded on sequencer for NGS.
  • DNA and RNA data are analyzed for DNA methylation and RNA expression respectively.

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  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des systèmes et des procédés permettant de diagnostiquer un état pathologique ou la progression d'une maladie comprenant la détection et la corrélation de changements de niveau de méthylation et d'expression de marqueurs du cancer, comprenant PD-L1, dans un échantillon de fluide biologique du patient. Les systèmes et les procédés sont applicables de manière similaire à la détection et à la corrélation de changements de niveau de méthylation et d'expression dans des tissus solides. Le présent procédé utilise un procédé de codage à barres pour l'analyse. Les systèmes et les procédés peuvent être utilisés pour prédire un cancer résistant à l'immunothérapie et pour surveiller une réponse thérapeutique.
PCT/US2018/025335 2016-04-15 2018-03-30 Systèmes et procédés de prédiction et de surveillance d'une thérapie anticancéreuse Ceased WO2018183796A1 (fr)

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US201762480278P 2017-03-31 2017-03-31
US62/480,278 2017-03-31
PCT/US2017/027830 WO2017181161A1 (fr) 2016-04-15 2017-04-16 Systèmes et procédés pour détecter des altérations génétiques
USPCT/US2017/027830 2017-04-16
USPCT/US2017/052832 2017-09-21
PCT/US2017/052832 WO2018057820A1 (fr) 2016-09-21 2017-09-21 Systèmes et procédés de détection combinée d'altérations génétiques

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US11702702B2 (en) 2016-04-15 2023-07-18 Predicine, Inc. Systems and methods for detecting genetic alterations
US11174503B2 (en) 2016-09-21 2021-11-16 Predicine, Inc. Systems and methods for combined detection of genetic alterations
EP3908657A4 (fr) * 2019-01-09 2022-09-14 Qiagen Sciences, LLC Procédés de détection d'analytes et compositions associées
CN110699435A (zh) * 2019-11-08 2020-01-17 宁波胤瑞生物医学仪器有限责任公司 一种用于pd-l1表达水平检测的试剂盒
WO2025007038A1 (fr) * 2023-06-30 2025-01-02 Guardant Health, Inc. Méthodes de détection précoce du cancer
WO2025106796A1 (fr) * 2023-11-15 2025-05-22 Guardant Health, Inc. Classification d'histologie du cancer du poumon non à petites cellules (cpnpc) au moyen de données de méthylation d'adn capturées à partir de biopsies liquides

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