[go: up one dir, main page]

WO2023044117A1 - Fluide de drainage pour diagnostic - Google Patents

Fluide de drainage pour diagnostic Download PDF

Info

Publication number
WO2023044117A1
WO2023044117A1 PCT/US2022/044010 US2022044010W WO2023044117A1 WO 2023044117 A1 WO2023044117 A1 WO 2023044117A1 US 2022044010 W US2022044010 W US 2022044010W WO 2023044117 A1 WO2023044117 A1 WO 2023044117A1
Authority
WO
WIPO (PCT)
Prior art keywords
fluid
tumor
variants
drain
drain fluid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2022/044010
Other languages
English (en)
Inventor
Jose P. ZEVALLOS
Aadel Chaudhuri
Stanley N. Lapidus
Theresa TRIBBLE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Washington University in St Louis WUSTL
Droplet Biosciences Inc
Original Assignee
Washington University in St Louis WUSTL
Droplet Biosciences Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Washington University in St Louis WUSTL, Droplet Biosciences Inc filed Critical Washington University in St Louis WUSTL
Priority to EP22870824.4A priority Critical patent/EP4405681A4/fr
Priority to CA3232274A priority patent/CA3232274A1/fr
Publication of WO2023044117A1 publication Critical patent/WO2023044117A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • C12Q2600/156—Polymorphic or mutational 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 related to diagnostic methods for identifying biomarkers indicative of disease.
  • Cancer is a leading cause of death globally. Early detection, while beneficial for most cancers, is often difficult. In part, this is because many cancers first develop without presenting any specific clinical symptoms, and diagnosis only occurs when the disease has reached a stage when it is difficult to treat.
  • Cancer detection has focused on liquid biopsy in blood or plasma for the detection of cell-free tumor DNA. Blood is of high clinical interest because of its accessibility. Unfortunately, many of these methods lack sensitivity. As a result, early cancer detection, when tumor DNA is present as only a minute fraction of the DNA collected from blood or plasma, is often difficult. Moreover, due to the lack of sensitivity, progression of the disease and its response to therapeutic intervention are difficult to monitor.
  • Tissue such as tumor tissue
  • Tissue samples are often difficult to access and subject to limited availability, especially without performing an invasive procedure.
  • cancer has spread or progressed.
  • the present invention provides methods for using drain fluid (i.e., effluent) obtained from medical procedures (e.g., medical interventions such as surgeries, biopsies, catheterizations, dissections, intubations and the like) to assess diagnostic biomarkers indicative of disease.
  • this “waste” fluid is actually a rich source of diagnostic information.
  • drain fluids comprising fluid originating in lymphatics and as interstitial fluid is more representative of the biomarker heterogeneity of a tumor than would be obtained from a biopsy sample.
  • biomarkers, such as nucleic acid variants or proteins, obtained from drain fluid are a more informative source of tumor genomics than would be apparent from a typical biopsy sample.
  • the information obtained from drain fluid is, therefore, informative for a diagnostic assay, whether performed on the obtained drain fluid or at a subsequent time in blood or downstream lymphatic fluid.
  • the invention provides both the ability to create a more informative personalized array of biomarkers as well as the ability to perform longitudinal monitoring of individual patients to assess recurrence, minimal residual disease and response to treatment generally.
  • drain fluid as a source of biomarkers indicative of disease.
  • the drain fluid can be obtained as effluent from a medical intervention, such as a surgery or biopsy. Drain fluid can also be obtained during treatment of a wound or interventional procedure. Methods taught herein provide sensitive and specific diagnostics that allow assessment of disease status, staging, and progression; as well as aiding in therapeutic selection and assessment of therapeutic efficacy.
  • drain fluid does not imply fluid obtained from a drain device, although that can be the case. Rather, drain fluid is intended to be fluid originating in the lymphatics and, in some cases, progressing via lymphatic channels to lymph glands and ultimately to blood.
  • Drain fluid may comprise interstitial fluid and may be obtained as a mixture with other bodily fluids, including blood.
  • a wash fluid or rinse may also be part of the drain fluid as obtained during a medical intervention.
  • the invention is focused on diagnostic content in the drain fluid or effluent originating in vivo.
  • drain fluid is a source of circulating tumor DNA (ctDNA), tumor cells, ratios of ctDNA to cells, mutations associated with cancer, oncogenes and the like.
  • ctDNA tumor DNA
  • the invention is also useful forthe assessment of diseases other than cancer, including infectious diseases, autoimmune diseases, endocrine diseases and the like.
  • drain fluid is lymphatic fluid obtained during a medical procedure.
  • the fluid may also be obtained as effluent derived from a medial procedure, such as, for example, fluid obtained from a colostomy bag (e.g., post bowel resection).
  • the fluid may also contain irrigation fluid, blood, plasma, particulates (including proteins) and cells. Accordingly, it may be necessary to isolate a fraction of interest from the drain fluid.
  • the invention contemplates that raw drain fluid contains a sufficient amount of diagnostic content that it can be used without any significant sample preparation.
  • the present invention provides methods for disease diagnosis comprising the steps of obtaining a biological fluid from a medical intervention, identifying in the fluid a biomarker indicative of disease, and diagnosing a disease or condition based on the presence of the biomarker in the fluid.
  • the disease can be any disease, including cancer, infectious disease, metabolic disease or an autoimmune disease.
  • the invention has particular application in the diagnosis of cancer.
  • Drain fluid for use in the invention preferably is surgical drain fluid, for example surgical fluid that would otherwise be discarded as medical waste.
  • Surgical drain fluid may comprise blood, plasma, aggregated tissue, irrigation fluid, lymphatic fluid, lymphovascular fluid, interstitial fluid or a combination thereof.
  • the fluid may further comprise bile, sweat, semen, vaginal secretions, stool, cerebrospinal fluid, synovial fluid, pleural fluid, peritoneal fluid, pericardial fluid, amniotic fluid, saliva, or mucus depending on the location from which it is obtained.
  • biomarkers indicative of disease in the drain fluid may be accomplished by any known method for biomarker detection.
  • biomarkers for disease may be detected by sequencing, for example protein or nucleic acid sequencing.
  • the biomarker may be DNA and/or RNA.
  • methods of the present invention contemplate creating a genomic profile from DNA or RNA in the drain fluid.
  • the profile maybe a patient’s germ line profile or may be of a tumor.
  • the biomarker detected (whether in drain fluid or subsequently in blood draws) has a lineage originating in cancerous or pre-cancerous tissue.
  • a nucleic acid variant detected in blood or drain fluid at one point in time may not have been present if detection had been performed earlier. That is due to the fact that tumor evolution may result in different variants being present at different stages of development.
  • biomarkers used for cancer detection according to the invention have their ultimate origin in a cancerous or pre-cancerous tissue.
  • Biomarkers for use in the invention may be any known biomarker for disease.
  • the biomarker may be a nucleic acid variant (DNA, any RNA species), a protein, any other molecule or compound, or a cell.
  • the origin of the biomarker may be an organism, for example a bacterium, a fungus, an animal cell, a tumor cell, a protozoan cell, or a virus.
  • the choice of specific biomarkers is dependent on their sensitivity and specificity of the biomarker for a particular condition, and also on technical aspects related to collection protocols, stability and detection from biological samples.
  • the biomarker may be one or more of interleukin- 1, interleukin-6, interleukin- 10, a tumor necrosis factor, matrix metalloproteinase- 1, matrix metalloproteinase-2, matrix metalloproteinase-9, matrix metalloproteinase-13, or a nucleic acid comprising a mutation.
  • Biomarkers are correlated with the diagnosis, prognosis, staging and development of disease; as well as therapeutic selection and response. In clinical oncology, early diagnosis and prognosis is critical for appropriate therapeutic intervention. For example, treatment protocols may be different if there are indications of metastatic disease in cancer. Accordingly, biomarkers detected in drain fluid may depend on the purpose for the assessment, e.g., to indicate normal biological processes, pathogenic processes, or pharmacologic response to therapeutic intervention.
  • methods of the invention contemplates the discovery that lymphatic fluid in surgical effluent frequently contains important biomarkers, often in greater abundance than in blood and plasma. Accordingly, methods of the invention may comprise the further step of separating lymphatic fluid from the overall drain fluid. Methods of the invention may also comprise assaying the surgical fluid without separating lymphatic fluid from the drain fluid.
  • the invention contemplates the detection of a ratio of circulating tumor cells to cell-free DNA and/or ratios of biomarkers.
  • the invention contemplates detecting an amount of a biomarker in drain fluid as compared to blood, plasma, or lymph node tissue from the same subject at the same time or at intervals over a period of time.
  • surgical drain fluid may comprise a greater ratio of circulating tumor cells to cell-free DNA than the same volume of blood or plasma. Without being bound to a single theory, this may be the result of large quantities of biomarker rich lymphatic fluid in interventional (e.g., surgical) waste.
  • the lymphatic fluid separated from, for example, a surgical effluent may comprise a greater volume of cells or nucleic acid molecules specific to a tumor than the same volume of blood obtained from the subject.
  • Drain fluid may be obtained by any known method.
  • the drain fluid may be obtained passively as the effluent from a medical procedure.
  • a catheter or a drain port for actively or passively collecting fluid.
  • Suction drainage for example using a vacuum, may also be used to obtain fluids during a surgical procedure.
  • the surgical drain fluid may be collected by using a syringe, pipet, or catheter, for example a Jackson-Pratt (JP) drain.
  • JP Jackson-Pratt
  • the drain fluid may be collected in or transferred to a container, for example a sample vessel, such as a vial, flask, or ampule, suitable forthe sterile collection of medical specimens.
  • Surgical fluid may also be collected from biohazard waste containers, for example a suction canister, filled during a procedure or diverted from a biohazard waste container during a surgical procedure.
  • Sample may be obtained by irrigating a surgical wound.
  • Irrigating fluid may comprise water, saline, antibiotic solutions, antiseptic agents, or a combination thereof.
  • Surgical drain fluid may be collected from any surgical procedure.
  • the surgery may comprise an open surgical procedure or an endoscopic procedure.
  • the surgical procedure may comprise an invasive procedure.
  • the surgical procedure may comprise a resection, biopsy, dissection, or excision.
  • the surgical procedure maybe a thoracentesis.
  • the surgical procedure may be a minimally invasive procedure, such as, for example, a stent placement.
  • the surgical procedure may be a procedure that is not a related to a disease being diagnosed.
  • the invention applies to any disease condition and the drain fluid may be from an unrelated interventional procedure.
  • Drain fluid may be obtained at any time during or following an interventional procedure.
  • drain fluid maybe collected at the time of intervention and then periodically over the course of hours, days or weeks.
  • the present invention also contemplates diagnosing disease by obtaining a representative sample of drain fluid and assaying the drain fluid for indicia of disease.
  • Obtaining a representative sample is especially important in cancer diagnosis because tumor-related biomarkers may not be present uniformly in the drain fluid, depending on volume, source of the drain fluid and collection techniques.
  • the drain fluid may be collected via a drain.
  • the drain maybe designed to be maintained at a wound site for a period of time after a surgical procedure.
  • cancer metastasis may be identified by identifying a cancer biomarker in drain fluid and determining whether the same biomarker is present in a lymph node or in blood. If the biomarker is found in the drain fluid but not in blood, it can be inferred that the tumor cells have yet reached the general circulation.
  • a DNA sample from a tumor may be obtained as well as a blood sample and surgical fluid. Each of the samples may be then tested for tumor DNA, and the ratios of tumor DNA in the tumor, lymphatic fluid, and blood may be used as an indicator of disease staging.
  • Methods of the invention also contemplate assessment of pharmaceutical efficacy.
  • accumulation oftumorDNA in drain fluid after therapy is indicative that the therapy is effective, as an increase in tumor DNA in drain fluid after therapy is indicative of the induction of cell death in the tumor.
  • real-time measurement during therapy is indicative of therapeutic efficacy.
  • the efficacy of systemic therapy is measure by the concentration of local biomarkers in the drain fluid.
  • the rate of accumulation or decrease of biomarkers in drain fluid is indicative of disease severity and whether a disease is progressing or regressing. For example, if drain fluid is measured at multiple time points, it is possible to calculate a slope of biomarker accumulation. The steepness of the slope is indicative of the velocity of change (either negative or positive). In the same way, the area under the curve resulting from multiple measurements in the drain fluid is indicative of disease progression or regression.
  • Tumor-informed plasma-based cfDNA assays for detection of minimal residual disease (MRD) after cancer treatment are limited by 1) sampling bias from a small sampling of the tumor, and 2) tumor heterogeneity and subclonality that develops during the course of carcinogenesis and metastasis.
  • surgical drain fluid in patients undergoing cancer surgery is enriched for tumor cfDNA that correlates with clinical outcome.
  • the characterization and/or quantification of tumor cfDNA in the surgical drain fluid is useful to measure locoregional minimal residual disease as well as for determining the risk of recurrence.
  • cfDNA in drain fluid broadly captures tumor heterogeneity in a manner that is difficult or impossible with the small biopsy sample typically obtained from primary tumor tissue.
  • drain fluid allows detection of a broader spectrum of variants, including high-risk variants, that represent the entirety of the tumor and lymph nodes metastases.
  • the invention provides a plasma-based minimum residual disease (MRD) surveillance assay, the content of which is informed by tumor-derived biomarkers (variants) detected in the primary tumor and/or in the drain fluid.
  • MRD plasma-based minimum residual disease
  • an embodiment of the invention comprises determining one or more tumor- derived biomarkers in drain fluid resulting from a medical intervention and preparing a panel for use in a blood-based assay to determine disease status.
  • the tumor derived biomarkers are selected from nucleic acids, proteins, carbohydrates, lipids and other biomolecules that are indicative of disease state.
  • the biomarkers are nucleic acids, and in particular, tumor-associated nucleic acid variants (i.e., mutations) that are obtained in drain fluid and are used to construct a panel of variant targets for a blood-based assay.
  • the blood-based assay (e.g., a plasma-based assay) is used to assess initial disease state or is used to assess disease state longitudinally in the patient.
  • the disease state is cancer and the blood-based assay panel is used to determine initial diagnosis, disease progression, response to treatment, metastasis, recurrence, and/or minimal residual disease.
  • a surgeon performs a tumor resection and collects drain fluid. Tumor DNA is then sequenced and informative or potentially -informative variants are determined. Variants in drain fluid cfDNA are also detected.
  • the drain fluid variants are useful to determine locoregional disease (including MRD) and to inform adjuvant therapy.
  • the resulting MRD panel for a blood-based assay is more informative of disease heterogeneity than a panel constructed solely from biopsy material.
  • an advantage of this aspect of the invention is that a panel constructed, at least in part, from cfDNA variants obtained in drain fluid allows for personalized surveillance monitoring of a patient.
  • variant panels based on drain fluid incorporates variants that may be missing in tumor biopsy which, by definition, may not be representative of overall tumor heterogeneity.
  • a biopsy may capture about 1mm of, for example, a 5cm tumor.
  • Drain fluid provides a more representative sample of the heterogeneity of a tumor, as drain fluid is not confined to only a portion of the tumor.
  • an assay panel based on drain fluid provides a representative and more informative assessment of tumor heterogeneity, which allows for more precision in patient longitudinal monitor.
  • Methods of the invention are also useful to construct a blood-based panel for assessment of therapeutic efficacy.
  • a panel created, at least in part, based on cfDNAin drain fluid is used to establish a baseline and then to monitor the patient through a course of treatment to determine the effect of the treatment on reducing the presence of tumor-associated cfDNA over time as a proxy for successful treatment of the primary tumor and/or mets.
  • Blood-based surveillance assays of the invention are also useful to inform therapeutic choice.
  • the invention contemplates creating a database of mutations identified in drain fluid (by specific mutation or type of mutation) and associated treatment outcomes.
  • the database is resident on computing system with transient memory and associated processors for storage of data and conducting one or more operations for processing inputted data in a manner designed and implemented by one or more programming routines.
  • Data from patient drain fluid is inputted into the computing system and analyzed for similarities with variants and known successful treatment outcomes in order to inform personalized treatment for the subject patient.
  • the computing system executes a supervised machine learning algorithm, such as a random forest algorithm, XGBoost algorithm, support vector machine learning algorithm, or a convoluted neural network, cyclic and acyclic graphing and other appropriate algorithms for supervised and/or unsupervised machine learning.
  • a supervised machine learning algorithm such as a random forest algorithm, XGBoost algorithm, support vector machine learning algorithm, or a convoluted neural network, cyclic and acyclic graphing and other appropriate algorithms for supervised and/or unsupervised machine learning.
  • drain fluid provides a more informative and representative assessment of tumor-derived variants that may present in blood or plasma.
  • methods of the invention are applicable to analysis of any nucleic acid variant (i.e., in DNA or any species of RNA) gene expression analysis, copy number variation, and/or epigenetic variants.
  • Variants can also be proteins, lipids, carbohydrates, and other biomolecules derived from a tumor. These biomarkers are present in lymphatic fluid and transit from a tumor via lymphatic channels to lymph glands and eventually to blood.
  • Obtaining a sample in drain fluid provides a representative sample that is proximal to the primary tumor (although one can perform the same analysis on a metastatic tumor or recurrent cancer) and thus provides a more representative sample as compared to a conventional tumor biopsy.
  • Fig. 1 is a schematic of a process of the invention for use in liquid biopsy.
  • the present invention provides methods for disease diagnosis comprising the steps of obtaining drain fluid from an interventional procedure, identifying in the fluid a biomarker indicative of disease, and diagnosing a disease or condition based on the presence of the biomarker in the fluid.
  • the present invention is based on the discovery that what has conventionally been considered, for example, surgical waste fluid, actually is a rich source of diagnostic content.
  • the lymphatic or lymph system is central to the body’s immune system.
  • the system contains lymphatic vessels that collect lymphatic fluid from peripheral tissues of the body and transport it to lymphatic ducts.
  • Lymph nodes of the system contain immune cells to fight infections and filter extra-cellular materials (e.g., cellular debris and byproducts, damaged cells, cancer cells, and pathogens) from the lymph fluid.
  • the right lymphatic duct and thoracic duct drain lymph fluid collected from the lymphatic system and return it to the bloodstream via the subclavian vain.
  • the lymph system is associated with many types of cancer and other pathologies.
  • cells of the lymph system itself such as in the lymph nodes, can be the source of cancers, such as lymphomas.
  • the lymph system can remove cancer cells (e.g., those the break away from a tumor) or other pathogens from a peripheral tissue of the body as part of its immune system functioning.
  • cancer cells attach to a portion of the immune system, including after collection, and begin to grow.
  • This metastasis is often exacerbated as the lymph system continues to circulate newly grown cancer cells throughout the system, and by extension, the body.
  • many cancers of the head, neck, breast, and glandular systems are associated with metastasis or other changes in the lymphatic system.
  • lymphatic system many surgical interventions involve the lymphatic system. This may include direct interventions of the lymphatic system, such as resection, dissection, or excision surgeries to remove a diseased portion of the lymphatic system or to obtain tissue samples. Additionally, given its role in the immune system, many surgical interventions, which do not directly target the lymphatic system, often require collecting and discarding lymphatic fluid, for example, during or after treatments to manage lymph fluid overload or facilitate wound healing. Often, as part of a postoperative regime, patients receive an implanted surgical drain, such as a JP drain, which removes lymph fluid that collects at the site of a surgery.
  • a JP drain which removes lymph fluid that collects at the site of a surgery.
  • the biomarker identified by the present invention may be any known biomarker for a given disease present in surgical fluid.
  • the biomarker may comprise tumor cells, immune cells, bacterial cells, viral host cells, donor organ cells, microvascular cells, cell-free DNA, cell-free RNA, circulating tumor DNA, messenger RNA, miRNA, exosomes, proteins, hormones, and other analytes.
  • the biomarker identified may depend on, for example, a specific patient, pathology, surgery type, and surgery site.
  • methods of the invention may provide diagnostic or prognostic information. For example, by identifying circulating tumor cells or cell-free tumor DNA, cancer may be diagnosed in the subject.
  • biomarkers may be identified and quantified using methods known in the art.
  • Suitable assays include, for example, nucleic acid sequencing, PCR, quantitative PCR, digital droplet PCR, Western blot target capture, proteomics, nucleic acid expression analysis, and antibody screening.
  • assays may include whole genome sequencing, next generation DNA sequencing, next generation RNA sequencing, multiplex PCR, methylation analysis, droplet PCR, droplet cell separation, or any combination thereof.
  • Fluorescent labels may be used to identify biomarkers.
  • a fluorescent label or fluorescent probe is a molecule that is attached chemically to aid in the detection of a biomarker. Fluorescent labeling generally uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the biomarker and can be attached chemically or biologically. Any known technique for fluorescent labeling may be used, for example enzymatic labeling, protein labeling, or genetic labeling. Any known fluorophore may also be used. Both the fluorophore and labelling technique may be selected and adjustedbasedon the biomarker to be identified. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.
  • Fluorescent labelling may be used to identify and quantify a biomarker in the surgical fluid sample without separating the components of the surgical fluid. For example, by providing fluorescent labels directly into the surgical fluid, fluorescent microscopy or a colorimetric assay can be used to identify and quantify the presence of the biomarker from a color change alone. For example, fluorescent labels may be applied to the surgical fluid in the surgical-suite during the surgical procedure to provide valuable information to the surgeon.
  • barcodes may be added to biomarker to aid in amplification, detection, or differentiation of the biomarker. Barcodes may be added to biomarkers by “tagging” the biomarker with the barcode. Tagging may be performed using any known method for barcode addition, for example direct ligation of barcodes to one or more of the ends of a nucleic acid molecule or protein. Nucleic acid molecules may, for example, be end repaired in order to allow for direct or blunt-ended ligation of the barcodes. Barcodes may also be added to nucleic acid molecules through first or second strand synthesis, for example using capture probes or primers. First and second strand synthesis is advantageously used in RNA analysis to generate tagged DNA molecules.
  • Unique molecular identifiers are a type of barcode that may be provided to biomarkers in a sample to make each biomarker, together with its barcode, unique, or nearly unique.
  • nucleic acid molecules this is accomplished by adding, e.g. by ligation or reverse transcription, one or more UMIs to each nucleic acid molecule such that it is unlikely that any two previously identical nucleic acid molecules, together with their UMIs, have the same sequence. By selecting an appropriate number of UMIs, every nucleic acid molecule in the sample, together with its UMI, will be unique or nearly unique.
  • One strategy for doing so is to provide to a sample of nucleic acid molecules a number of UMIs in excess of the number of starting nucleic acid molecules in the sample. By doing so, each starting nucleic molecule will be provided with different UMIs, therefore making each molecule together with its UMIs unique.
  • the number of UMIs provided may be as few as the number of identical nucleic acid molecules in the original sample. For example, where no more than six nucleic acid molecules in a sample are likely to be identical, as few as six different UMIs may be provided, regardless of the number of starting nucleic acid molecules.
  • UMIs are also advantageous in that they can be useful to correct for errors created during amplification, such as amplification bias or incorrect base pairing during amplification.
  • errors created during amplification such as amplification bias or incorrect base pairing during amplification.
  • UMIs because every nucleic acid molecule in a sample together with its UMI or UMIs is unique or nearly unique, after amplification and sequencing, molecules with identical sequences may be considered to refer to the same starting nucleic acid molecule, thereby reducing amplification bias.
  • Methods for error correction using UMIs are described in Karlsson et al., 2016, “Counting Molecules in cell-free DNA and single cells RNA”, Karolinska Institutet, Sweden, the contests of which are incorporated herein by reference.
  • sequencing may first comprise the step of preparing a cDNA library from barcoded RNA, for example through reverse transcription, and sequencing the cDNA.
  • cDNA sequencing may advantageously allow for the quantification of gene expression within the single cell, and can be useful to identify characteristics of the single cell to, for example, make a diagnosis, prognosis, or determine drug effectiveness.
  • Reverse transcription may be performed using without limitation dNTPs (mix of the nucleotides dATP, dCTP, dGTP and dTTP), buffer/s, detergent/s, or solvent/s, as required, and suitable enzyme such as polymerase or reverse transcriptase.
  • the polymerase used may be a DNA polymerase, and may be selected from Taq DNA polymerase, Phusion polymerase (as provided by Thermo Fisher Scientific, Waltham, Massachusetts), or Q5 polymerase.
  • Nucleic acid amplification reagents are commercially available, and maybe purchased from, for example, New England Biolabs, Ipswich, MA, USA.
  • the reverse transcriptase used in the presently disclosed targeted library preparation method may be for example, maxima reverse transcriptase.
  • the general parameters of the reverse transcription reaction comprise an incubation of about 15 minutes at 25 degrees and a subsequent incubation of about 90 minutes at 52 degrees.
  • Reverse transcription may be performed by oligos that have a free, 3 ’ poly-T region.
  • the 3 ’ portions of the cDNA capture oligos may include gene-specific sequences or oligomers, for example capture primers to reverse transcribe RNA guides comprising a capture sequence.
  • the oligomers may be random or “not-so-random” (NSR) oligomers (NSROs), such as random hexamers or NSR hexamers.
  • the oligos may include one or more handles such as primer binding sequences cognate to PCR primers that are used in the amplifying step or the sequences of NGS sequencing adaptors.
  • the reverse transcription primers may include template switching oligos (TSOs), which may include poly-G sequences that hybridize to and capture poly-C segments added during reverse transcription.
  • TSOs template switching oligos
  • Reverse transcription of non-polyadenylated RNA may comprise use of a capture sequence and a capture primer or probe.
  • Primer sequences may comprise a binding site, for example a primer sequence that would be expected to hybridize to a complementary sequence, if present, on any nucleic acid molecule released from a cell and provide an initiation site for a reaction.
  • the primer sequence may also be a “universal” primer sequence, i.e. a sequence that is complementary to nucleotide sequences that are very commonfor a particular set of nucleic acid fragments.
  • Primer sequences maybe P5 and P7 primers as provided by Illumina, Inc., San Diego, California.
  • the primer sequence may also allow a capture probe to bind to a solid support.
  • Reverse transcription can also be useful for adding a barcode or a UMI, or both to cDNA.
  • This process may comprise hybridizing the reverse transcription primer to the probe followed by a reverse transcription reaction.
  • the complement of a nucleic acid when aligned need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases.
  • Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, percent concentration of cytosine and guanine bases in the oligonucleotide, ionic strength, and incidence of mismatched base pairs.
  • Nucleic acid molecules may advantageously be amplified prior to sequencing.
  • Amplification may comprise methods for creating copies of nucleic acids by using thermal cycling to expose reactants to repeated cycles of heating and cooling, and to permit different temperature-dependent reactions (e.g. by Polymerase chain reaction (PCR). Any suitable PCR method known in the art may be used in connection with the presently described methods. Non limiting examples of PCR reactions include real-time PCR, nested PCR, multiplex PCR, quantitative PCR, or touchdown PCR.
  • Sequencing nucleic acid molecules may be performed by methods known in the art. For example, see, generally, Quail, et al., 2012, A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and IlluminaMiSeq sequencers, BMC Genomics 13 :341. Nucleic acid molecule sequencing techniques include classic dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, or preferably, next generation sequencing methods. For example, sequencing may be performed according to technologies described in U.S. Pub. 2011/0009278, U.S. Pub.
  • the conventional pipeline for processing sequencing data includes generating FASTQ- format files that contain reads sequenced from a next generation sequencing platform, aligning these reads to an annotated reference genome, and quantifying expression of genes.
  • These steps are routinely performedusingknown computer algorithms, which a person skilled in the art will recognize can be used for executing steps of the present invention. For example, seeKukurba, Cold Spring Harb Protoc, 2015 (11 ):951-969, incorporated by reference.
  • subjects After neck resection surgery to remove an oropharyngeal tumor, subjects receive an implanted surgical drain, e.g., a JP drain. 24 hours post-surgery, fluid is collected from a lymphatic duct from each subject via the drain. Each fluid sample is centrifuged and filtered. A nuclease, such as EDTA is added to each sample.
  • a JP drain e.g., a JP drain
  • Biomarkers associated with oropharyngeal cancer are isolated and measured from the samples, which include tumor-associated genetic material.
  • the tumor-associated genetic material includes, for example, one or more of cell-free nucleic acids, nucleic acids from a tumor, nucleic acids from an isolated exosome, and/or viral nucleic acids.
  • the tumor-associated genetic material is analyzed using one or more of nucleic acid sequencing, PCR, and/or Western blot.
  • results may include, for example, quantities of detected nucleic acids, mutations, variants, copy number, and expression patterns. These results may be compared with other bioassay results, either for other biomarkers in the fluid from the lymphatic duct and/or from a different sample type, such as blood or plasma.
  • one or more scores are produced indicative of the subjects’ conditions, disease states, and prognosis.
  • the scores provide a practitioner with valuable insight as to whether to pursue additional therapeutic intervention, e.g., additional surgery, medications, and active monitoring.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pathology (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • General Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Measuring And Recording Apparatus For Diagnosis (AREA)

Abstract

La présente invention concerne des procédés pour tester un fluide chirurgical à la recherche de biomarqueurs de maladie, notamment du cancer. Ce procédé comprend les étapes d'obtention du fluide chirurgical résultant d'une intervention médicale, d'identification du biomarqueur qui est révélateur d'une maladie dans le fluide chirurgical, et de diagnostic de ladite maladie sur la base de la présence dudit biomarqueur dans le fluide chirurgical. Le fluide chirurgical comprend un fluide lymphatique, un fluide lymphovasculaire ou une combinaison de ceux-ci et peut être obtenu à l'aide d'un cathéter ou d'un orifice de drainage.
PCT/US2022/044010 2021-09-20 2022-09-19 Fluide de drainage pour diagnostic Ceased WO2023044117A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP22870824.4A EP4405681A4 (fr) 2021-09-20 2022-09-19 Fluide de drainage pour diagnostic
CA3232274A CA3232274A1 (fr) 2021-09-20 2022-09-19 Fluide de drainage pour diagnostic

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202163246253P 2021-09-20 2021-09-20
US63/246,253 2021-09-20
US202163282449P 2021-11-23 2021-11-23
US63/282,449 2021-11-23

Publications (1)

Publication Number Publication Date
WO2023044117A1 true WO2023044117A1 (fr) 2023-03-23

Family

ID=85603543

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2022/044010 Ceased WO2023044117A1 (fr) 2021-09-20 2022-09-19 Fluide de drainage pour diagnostic

Country Status (3)

Country Link
EP (1) EP4405681A4 (fr)
CA (1) CA3232274A1 (fr)
WO (1) WO2023044117A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024020527A1 (fr) * 2022-07-22 2024-01-25 Droplet Biosciences, Inc. Effluent pour dosages de culture cellulaire
WO2024130189A1 (fr) * 2022-12-16 2024-06-20 Droplet Biosciences, Inc. Collecte de cellules proximales de tumeur

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160033511A1 (en) * 2013-03-13 2016-02-04 Creatics Llc Methods and compositions for detecting pancreatic cancer
US20170211153A1 (en) * 2016-01-22 2017-07-27 Mayo Foundation for Medical Education and Research, a charitable corporation of Minnesota Algorithmic Approach for Determining the Plasma Genome Abnormality PGA and the Urine genome abnormality UGA scores based on Cell Free cfDNA Copy Number Variations in Plasma and Urine.
US20170276674A1 (en) * 2011-01-13 2017-09-28 Sarah Hamm-Alvarez Bioassay for the early detection of autoimmune diseases
WO2021127065A1 (fr) * 2019-12-16 2021-06-24 Washington University Procédé de mesure d'adn acellulaire dans un fluide de drainage chirurgical pour sélectionner une thérapie adjuvante

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170276674A1 (en) * 2011-01-13 2017-09-28 Sarah Hamm-Alvarez Bioassay for the early detection of autoimmune diseases
US20160033511A1 (en) * 2013-03-13 2016-02-04 Creatics Llc Methods and compositions for detecting pancreatic cancer
US20170211153A1 (en) * 2016-01-22 2017-07-27 Mayo Foundation for Medical Education and Research, a charitable corporation of Minnesota Algorithmic Approach for Determining the Plasma Genome Abnormality PGA and the Urine genome abnormality UGA scores based on Cell Free cfDNA Copy Number Variations in Plasma and Urine.
WO2021127065A1 (fr) * 2019-12-16 2021-06-24 Washington University Procédé de mesure d'adn acellulaire dans un fluide de drainage chirurgical pour sélectionner une thérapie adjuvante

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP4405681A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024020527A1 (fr) * 2022-07-22 2024-01-25 Droplet Biosciences, Inc. Effluent pour dosages de culture cellulaire
US12416626B2 (en) 2022-07-22 2025-09-16 Droplet Biosciences, Inc. Methods for evaluating candidate therapy efficacy based on changes in lymphatic fluid biomarkers
WO2024130189A1 (fr) * 2022-12-16 2024-06-20 Droplet Biosciences, Inc. Collecte de cellules proximales de tumeur

Also Published As

Publication number Publication date
EP4405681A1 (fr) 2024-07-31
CA3232274A1 (fr) 2023-03-23
EP4405681A4 (fr) 2025-08-06

Similar Documents

Publication Publication Date Title
US20180068058A1 (en) Methods and compositions for sample identification
WO2023044117A1 (fr) Fluide de drainage pour diagnostic
US20250084488A1 (en) Drain fluid for diagnostics
CN108796069A (zh) 心肌梗死的诊断标志物-ing1基因
WO2024173105A1 (fr) Fluides de drainage pour le diagnostic et la surveillance de maladies
CN108624680B (zh) Rae1基因或蛋白作为诊断心肌梗死的生物标志物的应用
CN117625777A (zh) 外泌体miRNA-365a-5p作为分子标志物的应用
CN118360386A (zh) 一种基于C15orf39基因的系统性红斑狼疮诊断试剂盒
US20240094213A1 (en) Thoracic duct as a sample collection reservoir
CN116814793A (zh) 用于乳腺癌诊断和/或预后诊断的标志物及其应用
WO2023021978A1 (fr) Méthode d'examen d'une maladie auto-immune
CN117316281A (zh) 基于cfDNA在TSS附近测序覆盖深度特征的癌症无创早筛方法
US12157920B1 (en) Methods for diagnosing and assessing disease using drain fluid
CA3233049A1 (fr) Fluide lymphatique pour diagnostic
CN113817818B (zh) 用于诊断过敏性气道炎症的工具
CN113817817B (zh) 一种诊断过敏性气道炎症的方法
US20230123183A1 (en) Lymphatic fluid for diagnostics
CN112410434A (zh) 一种血浆circRNA标志物及其应用
EP4665876A1 (fr) Fluides de drainage pour le diagnostic et la surveillance de maladies
CN117316280B (zh) 一种基于cfDNA末端序列特征的癌症无创早筛方法及系统
US11676724B1 (en) Method of calculating diagnostic score for prostate cancer and use thereof
JP7748744B2 (ja) 関節リウマチを検査する方法
US20230127134A1 (en) Methods for cancer treatment
CN108753957A (zh) 诊断心肌梗死的ivd基因及其应用
CN108796067A (zh) 血液中maea基因的诊断新功能

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22870824

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 3232274

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2022870824

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022870824

Country of ref document: EP

Effective date: 20240422