[go: up one dir, main page]

WO2018174859A1 - Procédés et compositions pour la détection d'un carcinome pulmonaire à cellules squameuses à un stade précoce à l'aide d'un profilage d'expression de rnaseq - Google Patents

Procédés et compositions pour la détection d'un carcinome pulmonaire à cellules squameuses à un stade précoce à l'aide d'un profilage d'expression de rnaseq Download PDF

Info

Publication number
WO2018174859A1
WO2018174859A1 PCT/US2017/023473 US2017023473W WO2018174859A1 WO 2018174859 A1 WO2018174859 A1 WO 2018174859A1 US 2017023473 W US2017023473 W US 2017023473W WO 2018174859 A1 WO2018174859 A1 WO 2018174859A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell carcinoma
squamous cell
lung squamous
reagents
sample
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/US2017/023473
Other languages
English (en)
Inventor
Bruce Xuefeng Ling
Limin Chen
Shiying Hao
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.)
Mprobe Inc
Original Assignee
Mprobe 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 Mprobe Inc filed Critical Mprobe Inc
Priority to PCT/US2017/023473 priority Critical patent/WO2018174859A1/fr
Publication of WO2018174859A1 publication Critical patent/WO2018174859A1/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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • 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
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • NSCLC non-small-ceil lung cancer
  • SCCs squamous cell carcinomas
  • RNA-seq technology provides a revolutionary tool for transcriptome analysis. Compared with microarray platform, RNA-seq has less background noise due to image analysis and is more sensitive in detection of transcripts with low-abundance or higher fold change in expression. In this invention, we use RNA-seq to find biomarkers for iung squamous cell carcinoma early detection.
  • methods are provided for detecting the level of at least one, at least two, at least three, at least four, or ali of the target moiecuies selected from Table 2, or any sub-combinations thereof, in a sample from a subject.
  • methods are provided for detecting the level of at least one, at least two, at least three, at least four, or all early stage lung squamous cell carcinoma biomarkers identified in experiment conducted during development of embodiments of the present invention.
  • biomarkers are selected from Table 2, or any subcombinations thereof.
  • a method comprises detecting the level of one or more biomarkers in a sample from a subject.
  • a method of monitoring lung squamous cell carcinoma (e.g., response to treatment, likelihood of mortality, etc.) in a subject comprises forming a biomarker panel having 50 biomarker proteins from lung squamous cell carcinoma biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., comprising DLX6, CACNA2D2, GAB R A3, CLDN18, PITX1 , FAT2, SUSD2, CLIC5, COL7A1 , TNNC1 , HOXC13, DUSP9, CNTN6, DSG3, DSC3, ADAMTS8, GPR87, FCN3, ABCA12, GGTLC1 , GP 6A, CERS3, GRIA1 , GRHL3, CD300LG, VEGFD, T EM100, CA4, GPD1, FAM83B, FA 107A, CALML3, NXPH4, CEMP1 , GKN2, C12orf56, KRT16, AGER, KRT6
  • N is 1 to 50. In some embodiments, N is 2 to 50. In some embodiments, methods comprise panels of any combination of the lung squamous cell carcinoma biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., DLX6, CACNA2D2, GABRA3, CLDN18, PITX1 , FAT2, SUSD2, CLIC5, COL7A1 , TNNC1 , HOXC13, DUSP9, CNTN6, DSG3, DSC3, ADAMTS8, GPR87, FCN3, ABCA12, GGTLC1 , GPM6A, CERS3, GRIA1 , GRHL3, CD300LG, VEGFD, TME 100, CA4, GPD1 , FA 83B, FAM107A, CALML3, NXPH4, MCEMP1 , GKN2, C12orf56, KRT16, AGER, KRT6A, SERPINB5, SFTA1P, AC098973.2, KCN B2-AS
  • methods comprise comparing biomarker(s) level to a reference value/range or a threshold. In some embodiments, deviation of the biomarker(s) level from the reference value/range, or exceeding or failing to meet the threshold, is indicative of a diagnosis, prognosis, etc. for the subject.
  • each biomarker may be a protein biomarker.
  • the method may comprise contacting biomarkers of the sample from the subject with a set of biomarker capture reagents, wherein each biomarker capture reagent of the set of biomarker capture reagents specifically binds to a biomarker being detected.
  • each biomarker capture reagent of the set of biomarker capture reagents specifically binds to a different biomarker being detected.
  • each biomarker capture reagent may be an antibody or an aptamer.
  • a biomarker is an RNA transcript.
  • the method may comprise contacting biomarkers of the sample from the subject with a set of biomarker capture reagents, wherein each biomarker capture reagent of the set of biomarker capture reagents specifically binds to a biomarker being detected.
  • each biomarker capture reagent of the set of biomarker capture reagents specifically binds to a different biomarker being detected.
  • each biomarker capture reagent may be a nucleic acid probe.
  • the sample may be a biological sample (e.g., tissue, fluid (e.g., blood, urine, saliva, etc.), etc.).
  • the sample is filtered, concentrated (e.g., 2-fold, 5-fold, 10 fold, 20-fold, 50-fold, 100-fold, or more), diluted, or un-manipulated.
  • a methods further comprise treating the subject for lung squamous cell carcinoma.
  • treating the subject for lung squamous cell carcinoma comprises a treatment regimen of administering one or more chemotherapeutic, radiation, surgery, etc.
  • biomarkers described herein are monitored before, during, and/or after treatment.
  • methods comprise providing palliative treatment (e.g., symptom relief) to a subject suffering from lung squamous cell carcinoma, but not providing interventional treatment of the lung squamous cell carcinoma.
  • palliative treatment e.g., symptom relief
  • methods comprise providing palliative treatment (e.g., symptom relief) to a subject suffering from lung squamous cell carcinoma, but not providing interventional treatment of the lung squamous cell carcinoma.
  • palliative care is pursued in place of lung treatment.
  • palliative care is provided in addition to treatment for lung squamous cell carcinoma.
  • a method comprises detecting the level of one or more lung squamous cell carcinoma biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., DLX6, CACNA2D2, GABRA3, CLDN18, PITX1 , FAT2, SUSD2, CLIC5, COL7A1 , TNNC1 , HOXC13, DUSP9, CNTN6, DSG3, DSC3, ADAMTS8, GPR87, FCN3, ABCA12, GGTLC1 , GPM6A, CERS3, GRIA1 , GRHL3, CD300LG, VEGFD, TMEM100, CA4, GPD1 , FAM83B, FAM107A, CALML3, NXPH4, MCEMP1 , GKN2, C12orf56, KRT16, AGER, KRT6A, SERPI
  • the method further comprises measuring the level one or more of the biomarkers at a second time point.
  • lung squamous cell carcinoma severity is improving (e.g., declining) if the level of said biomarkers improved at the second time point than at the first time point.
  • biomarkers or panels thereof provide a prognosis regarding the future course lung squamous cell carcinoma in a subject (e.g., likelihood of survival, likelihood of mortality, likelihood of response to therapy, etc.).
  • treatment decisions e.g., whether to treat, surgery, radiation, chemotherapy, etc.
  • kits are provided.
  • a kit comprises at least one, at least two, at least three, at least four, of at least five capture/detection reagents (e.g., antibody, probe, etc.), wherein each capture/detection reagents specifically binds to a different biomarker (e.g., protein or nucleic acid) selected from the lung squamous cell carcinoma biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., DLX6, CACNA2D2, GABRA3, CLDN18, PITX1 , FAT2, SUSD2, CLIC5, COL7A1 , TNNC1 , HOXC13, DUSP9, CNTN6, DSG3, DSC3, ADAMTS8, GPR87, FCN3, ABCA12, GGTLC1 , GPM6A, CERS3, GRIA1 , GRHL3, CD300LG, VEGFD, TMEM100, CA4, GPD1
  • a different biomarker
  • N is 1 to 50. In some embodiments, N is 2 to 50. In some embodiments, N is 3 to 50. In some embodiments, N is 4 to 50. In some
  • N is 5 to 50.
  • at least one of the 50 biomarker proteins is selected from the lung squamous cell carcinoma biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., DLX6,
  • compositions comprising proteins of a sample from a subject and at
  • a different biomarker selected from the lung squamous cell carcinoma biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., DLX6, CACNA2D2, GABRA3, CLDN18, PITX1 , FAT2, SUSD2, CLIC5, COL7A1 , TNNC1, HOXC13, DUSP9, CNTN6, DSG3, DSC3, ADAMTS8, GPR87, FCN3, ABCA12, GGTLC1 , GPM6A, CERS3, GRIA1 , GRHL3, CD300LG, VEGFD, TMEM100, CA4, GPD1 , FAM83B, FAM107A, CALML3, NXPH4, MCEMP1 , GKN2, C12orf56, KRT16, AGER, KRT6A, SERPINB5, SFTA1 P, AC098973.2, KCN B2-AS1 , INMT, ECEL
  • FIG. 1 The analysis procedure of RNA sequencing data. Each step and packages used in alignment, quantification, and DE analysis are described in this figure.
  • Figure 2 Scatterplot of calculated probabilities of lung squamous cell carcinoma with selected 50 gene panel.
  • the model was trained with Random Forest algorithm, 366/562 case/control (407/624 in total) were selected out randomly to train the model.
  • Figure 4 Unsupervised hierarchical duster analysis with heat map showing the abundance pattern of selected biomarkers of early stage lung squamous cell carcinoma patients versus normal subjects.
  • lung squamous cell carcinoma biomarkers are provided.
  • a “biomarker” or “marker” it is meant a molecular entity whose representation in a sample is associated with a disease phenotype.
  • lung squamous cell carcinoma it is meant a subtype of non-small cell lung cancer that is often diagnosed in an outer area of the lung and arises from the secretory (glandular) cells located in the epithelium lining the bronchi.
  • a lung squamous cell carcinoma “biomarker” or “lung squamous cell carcinoma marker” it is meant a molecular entity whose representation in a sample is associated with a lung squamous cell carcinoma phenotype, e.g., the presence of lung squamous cell carcinoma, the stage of lung squamous cell carcinoma, a prognosis associated with the lung squamous cell carcinoma, the predictability of the lung squamous cell carcinoma being responsive to a therapy, etc.
  • the marker may be said to be differentially represented in a sample having a lung squamous cell carcinoma phenotype.
  • Lung squamous cell carcinoma biomarkers include proteins that are differentially represented in a lung squamous cell carcinoma phenotype and their corresponding genetic sequences, i.e., mRNA, DNA, etc.
  • a “gene” or “recombinant gene” it is meant a nucleic acid comprising an open reading frame that encodes for the protein. The boundaries of a coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence.
  • a gene may optionally include its natural promoter (i.e., the promoter with which the exons and introns of the gene are operabiy linked in a non-recombinant cell , i.e., a naturally occurring cell), and associated regulatory sequences, and may or may not have sequences upstream of the AUG start site, and may or may not include untranslated leader sequences, signal sequences, downstream untranslated sequences, transcriptional start and stop sequences, polyadenylation signals, translational start and stop sequences, ribosome binding sites, and the like.
  • its natural promoter i.e., the promoter with which the exons and introns of the gene are operabiy linked in a non-recombinant cell , i.e., a naturally occurring cell
  • associated regulatory sequences may or may not have sequences upstream of the AUG start site, and may or may not include untranslated leader sequences, signal sequences, downstream untranslated sequences, transcriptional start and stop sequences
  • gene product or "expression product” are used herein to refer to the RNA transcription products (transcripts) of the gene, including mRNA; and the polypeptide translation products of such RNA transcripts, i.e. the amino acid product encoded by a gene.
  • a gene product can be, for example, an RNA transcript of the gene, e.g. an unspliced RNA, an mRNA, a splice variant mRNA, a microRNA, a fragmented RNA, etc.; or an amino acid product encoded by the gene, including, for example, full length polypeptide, splice variants of the full length polypeptide, post-translationally modified polypeptide, and fragments of the gene product, e.g.
  • an elevated level of marker or marker activity may be associated with the lung squamous cell carcinoma phenotype.
  • a reduced level of marker or marker activity may be associated with the lung squamous cell carcinoma phenotype.
  • T is used to categorize the pathology of the tumor(TX: Primary tumor cannot be assessed, or tumor proven by the presence of malignant cells in sputum or bronchial washings but not visualized by imaging or bronchoscopy; TO: No evidence of primary tumor; Tis Carcinoma in situ:Tis (AIS): adenocarcinoma, ⁇ (SCIS):
  • T1 Tumor 3 cm or less in greatest dimension, surrounded by lung or visceral pleura, without bronchoscopic evidence of invasion more proximal than the lobar bronchus (ie, not in the main bronchus); the uncommon superficial spreading tumor of any size with its invasive component limited to the bronchial wall, which may extend proximal to the main bronchus, is also classified as T1a,T1mi: Minimally invasive adenocarcinoma;T1a: Tumor 1 cm or less in greatest dimension;T1b: Tumor more than 1 cm but not more than 2 cm in greatest dimension;T1c: Tumor more than 2 cm but not more than 3 cm in greatest dimension; T2:
  • Tumor more than 3 cm but not more than 5 cm; or tumor with any of the following features (T2 tumors with these features are classified T2a if 4 cm or less or if size cannot be determined and as T2b if greater than 4 cm but not larger than 5 cm): Involves main bronchus regardless of distance to the carina, but without involving the carina; Invades visceral pleura; Associated with atelectasis or obstructive pneumonitis that extends to the hilar region, either involving part of the lung or the entire lung; T2a: Tumor more than 3 cm but not more than 4 cm in greatest dimension; T2b:Tumor more than 4 cm but not more than 5 cm in greatest dimension; T3:
  • T1mi minimally invasive adenocarcinoma
  • Tis tumor in situ.
  • Detection of Biomarkers and Determination of Biomarker Levels The presence of a biomarker or a biomarker level for the biomarkers described herein can be detected using any of a variety of analytical methods.
  • a biomarker level is detected using a capture reagent.
  • the capture reagent contains a feature that is reactive with a secondary feature on a solid support.
  • the capture reagent is exposed to the biomarker in solution, and then the feature on the capture reagent is used in conjunction with the secondary feature on the solid support to immobilize the biomarker on the solid support.
  • the capture reagent is selected based on the type of analysis to be conducted.
  • Capture reagents include but are not limited to aptamers, antibodies, other antibody mimetics and other protein scaffolds, autoantibodies, chimeras, small molecules, F(ab')2 fragments, single chain antibody fragments, FV fragments, single chain FV fragments, nucleic acids, lectins, ligand-binding receptors, affybodies, nanobodies, imprinted polymers, avimers, peptidomimetics, hormone receptors, cytokine receptors, and synthetic receptors, and modifications and fragments of these.
  • biomarker presence or level is detected using a
  • biomarker/capture reagent complex the biomarker presence or level is derived from the biomarker/capture reagent complex and is detected indirectly, such as, for example, as a result of a reaction that is subsequent to the biomarker/capture reagent interaction, but is dependent on the formation of the biomarker/capture reagent complex.
  • biomarker presence or level is detected directly from the biomarker in a biological sample.
  • biomarkers are detected using a multiplexed format that allows for the simultaneous detection of two or more biomarkers in a biological sample.
  • capture reagents are immobilized, directly or indirectly, covalently or non-covalently, in discrete locations on a solid support.
  • a multiplexed format uses discrete solid supports where each solid support has a unique capture reagent associated with that solid support, such as, for example quantum dots.
  • an individual device is used for the detection of each one of multiple biomarkers to be detected in a biological sample. Individual devices are configured to permit each biomarker in the biological sample to be processed simultaneously.
  • the fluorescent label is a fluorescent dye molecule.
  • the fluorescent dye molecule includes at least one substituted indolium ring system in which the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance, in some embodiments, the dye molecule includes an AlexFluor molecule, such as, for example, AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor680, or AlexaFluor 700.
  • the dye molecule includes a first type and a second type of dye molecule, such as, e.g., two different AlexaFluor molecules. In some embodiments, the dye molecule includes a first type and a second type of dye molecule, and the two dye molecules have different emission spectra.
  • Fluorescence can be measured with a variety of instrumentation compatible with a wide range of assay formats.
  • instrumentation for example, spectrofluorimeters have been designed to analyze microtiter plates, microscope slides, printed arrays, cuvettes, etc. See Principles of
  • a chemiluminescence tag is optionally used to label a component of the biomarker/capture complex to enable the detection of a biomarker level.
  • Suitable chemiluminescent materials include any of oxalylchloride, Rodamin 6G, Ru(bipy)32+, TMAE (tetrakis(dimethyiamino)ethylene), Pyrogallol (1 ,2,3-trihydroxibenzene), Lucigenin, peroxyoxalates, Aryl oxalates, Acridinium esters, dioxetanes, and others.
  • the detection method includes an enzyme/substrate combination that generates a detectable signal that corresponds to the biomarker level (e.g., using the techniques of ELISA, Western blotting, isoelectric focusing).
  • the enzyme catalyzes a chemical alteration of the chromogenic substrate which can be measured using various techniques, including spectrophotometry, fluorescence, and chemiluminescence.
  • Suitable enzymes include, for example, luciferases, luciferin, malate dehydrogenase, urease, horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, uricase, xanthine oxidase, lactoperoxidase, microperoxidase, and the like.
  • HRPO horseradish peroxidase
  • alkaline phosphatase beta-galactosidase
  • glucoamylase lysozyme
  • glucose oxidase galactose oxidase
  • glucose-6-phosphate dehydrogenase uricase
  • xanthine oxidase lactoperoxidase
  • microperoxidase and the like.
  • the detection method is a combination of fluorescence, chemiluminescence, radionuclide or enzyme/substrate combinations that generate a
  • multimodal signaling has unique and advantageous characteristics in biomarker assay formats.
  • the biomarker levels for the biomarkers described herein is detected using any analytical methods including, singleplex aptamer assays, multiplexed aptamer assays, singleplex or multiplexed immunoassays, mRNA expression profiling histological/cytological methods, etc. as discussed below.
  • Measuring mRNA in a biological sample may, in some embodiments, be used as a surrogate for detection of the level of a corresponding protein in the biological sample.
  • a biomarker or biomarker panel described herein can be detected by detecting the appropriate RNA.
  • mRNA expression levels are measured by reverse transcription quantitative polymerase chain reaction (RT-PCR followed with qPCR).
  • RT-PCR reverse transcription quantitative polymerase chain reaction
  • qPCR reverse transcription quantitative polymerase chain reaction
  • qPCR fluorescence as the DNA amplification process progresses.
  • qPCR can produce an absolute measurement such as number of copies of mRNA per ceil.
  • Northern blots, microarrays, RNAseq, Invader assays, and RT-PCR combined with capillary electrophoresis have all been used to measure expression levels of mRNA in a sample. See Gene Expression Profiling; Methods and Protocois, Richard A. Shimkets, editor, Humana Press, 2004; herein incorporated by reference in its entirety.
  • Immunoassay methods are based on the reaction of an antibody to its corresponding target or analyte and can detect the analyte in a sample depending on the specific assay format.
  • monoclonal antibodies and fragments, thereof are often used because of their specific epitope recognition.
  • Polyclonal antibodies have also been successfully used in various immunoassays because of their increased affinity for the target as compared to monoclonal antibodies.
  • Immunoassays have been designed for use with a wide range of biological sample matrices. Immunoassay formats have been designed to provide qualitative, semi-quantitative, and quantitative results.
  • Quantitative results are generated through the use of a standard curve created with known concentrations of the specificanalyte to be detected.
  • the response or signal from an unknown sample is plotted onto the standard curve, and a quantity or level corresponding to the target in the unknown sample is established.
  • Numerous immunoassay formats have been designed.
  • ELISA or EIA can be quantitative for the detection of an analyte. This method relies on attachment of a label to either the analyte or the antibody and the label component includes, either directly orindirectly, an enzyme.
  • ELISA tests may be formatted for direct, indirect, competitive, or sandwich detection of the analyte. Other methods rely on labels such as, for example, radioisotopes (I 125 ) or fluorescence.
  • Additional techniques include, for example, agglutination, nephelometry, turbidimetry, Western blot, immunoprecipitation, immunocytochemistry, immunohistochemistry, flow cytometry, Luminex assay, and others (see ImmunoAssay: A Practical Guide, edited by Brian Law, published by Taylor & Francis, Ltd., 2005 edition; herein incorporated by reference in its entirety).
  • Exemplary assay formats include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, fluorescent, chemiluminescence, and fluorescence resonance energy transfer (FRET) or time resolved-FRET (TR-FRET) immunoassays.
  • ELISA enzyme-linked immunosorbent assay
  • FRET fluorescence resonance energy transfer
  • TR-FRET time resolved-FRET
  • biomarkers include biomarker immunoprecipitation followed by quantitative methods that allow size and peptide level discrimination, such as gel electrophoresis, capillary electrophoresis, planar electrochromatography, and the like.
  • Methods of detecting and/or for quantifying a detectable label or signal generating material depend on the nature of the label.
  • the products of reactions catalyzed by appropriate enzymes can be, without limitation, fluorescent, luminescent, or radioactive or they may absorb visible or ultraviolet light.
  • detectors suitable for detecting such detectable labels include, without limitation, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and densitometers.
  • Any of the methods for detection can be performed in any format that allows for any suitable preparation, processing, and analysis of the reactions. This can be, for example, in multi-well assay plates (e.g., 96 wells or 384 wells) or using any suitable array or microarray. Stock solutions for various agents can be made manually or robotically, and all subsequent pipetting, diluting, mixing, distribution, washing, incubating, sample readout, data collection and analysis can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting a detectable label.
  • the biomarkers described herein may be detected in a variety of tissue samples using histological or cytological methods.
  • one or more capture reagent/s specific to the corresponding biomarkers are used in a cytological evaluation of a sample and may include one or more of the following: collecting a cell sample, fixing the cell sample, dehydrating, clearing, immobilizing the cell sample on a microscope slide,
  • the cell sample is produced from a cell block.
  • one or more capture reagent/s specific to the corresponding biomarkers are used in a histological evaluation of a tissue sample and may include one or more of the following: collecting a tissue specimen, fixing the tissue sample, dehydrating, clearing, immobilizing the tissue sample on a microscope slide, permeabilizing the tissue sample, treating for analyte retrieval, staining, destaining, washing, blocking, rehydrating, and reacting with capture reagent/s in a buffered solution.
  • fixing and dehydrating are replaced with freezing.
  • results are analyzed and/or reported (e.g., to a patient, clinician, researcher, investigator, etc.).
  • Results, analyses, and/or data e.g., signature, disease score, diagnosis, recommended course, etc. are identified and/or reported as an
  • a result may be produced by receiving or generating data
  • results determined by methods described herein can be independently verified by further or repeat testing.
  • analysis results are reported (e.g., to a health care professional (e.g., laboratory technician or manager, physician, nurse, or assistant, etc.), patient, researcher, investigator, etc.).
  • a result is provided on a peripheral, device, or component of an apparatus.
  • an outcome is provided by a printer or display.
  • an outcome is reported in the form of a report.
  • an outcome can be displayed in a suitable format that facilitates downstream use of the reported information.
  • Generating and reporting results from the methods described herein comprises transformation of biological data (e.g., presence or level of biomarkers) into a representation of the characteristics of a subject (e.g., likelihood of mortality, likelihood corresponding to treatment, etc.). Such a representation reflects information not determinable in the absence of the method steps described herein. Converting biologic data into understandable characteristics of a subject allows actions to be taken in response such information.
  • a downstream individual upon receiving or reviewing a report comprising one or more results determined from the analyses provided herein, will take specific steps or actions in response. For example, a decision about whether or not to treat the subject, and/or how to treat the subject is made.
  • receiving a report refers to obtaining, by a communication means, a written and/or graphical representation comprising results or outcomes of analysis.
  • the report may be generated by a computer or by human data entry, and can be communicated using electronic means (e.g., over the internet, via computer, via fax, from one network location to another location at the same or different physical sites), or by another method of sending or receiving data (e.g., mail service, courier service and the like).
  • the outcome is transmitted in a suitable medium, including, without limitation, in verbal, document, or file form.
  • the file may be, for example, but not limited to, an auditory file, a computer readable file, a paper file, a laboratory file or a medical record file.
  • a report may be encrypted to prevent unauthorized viewing.
  • systems and method described herein transform data from one form into another form (e.g., from biomarker levels to diagnoistic/prognostic determination, etc.).
  • the terms “transformed”, “transformation”, and grammatical derivations or equivalents thereof refer to an alteration of data from a physical starting material (e.g., biological sample, etc.) into a digital representation of the physical starting material (e.g., biomarker levels), a condensation/representation of that starting material (e.g., risk level), or a recommended action (e.g., treatment, no treatment, etc.).
  • kits can contain one or more detectable labels as described herein, such as a fluorescent moiety, etc.
  • a kit includes (a) one or more capture reagents for detecting one or more biomarkers in a biological sample, and optionally (b) one or more software or computer program products for providing a diagnosis/prognosis for the individual from whom the biological sample was obtained. Aiternatively, rather than one or more computer program products, one or more instructions for manually performing the above steps by a human can be provided.
  • a kit comprises a solid support, a capture reagent, and a signal generating material.
  • the kit can also include instructions for using the devices and reagents, handling the sample, and analyzing the data. Further the kit may be used with a computer system or software to analyze and report the result of the analysis of the biological sample.
  • kits can also contain one or more reagents (e.g., solubilization buffers, detergents, washes, or buffers) for processing a biological sample.
  • reagents e.g., solubilization buffers, detergents, washes, or buffers
  • Any of the kits described herein can also include, e.g., buffers, blocking agents, mass spectrometry matrix materials, serum/plasma separators, antibody capture agents, positive control samples, negative control samples, software and information such as protocols, guidance and reference data.
  • kits are provided for the analysis of glioma, wherein the kits comprise PCR primers for one or more biomarkers described herein.
  • a kit may further include instructions for use and correlation of the biomarkers.
  • kits may include a DNA array containing the complement of one or more of the biomarkers described herein, reagents, and/or enzymes for amplifying or isolating sample DNA.
  • the kits may include reagents for real-time PCR, for example, TaqMan probes and/or primers, and enzymes.
  • a kit can comprise (a) reagents comprising at least one capture reagent for determining the level of one or more biomarkers in a test sample, and optionally (b) one or more algorithms or computer programs for performing the steps of comparing the amount of each biomarker quantified in the test sample to one or more predetermined cutoffs.
  • an algorithm or computer program assigns a score for each biomarker quantified based on said comparison and, in some embodiments, combines the assigned scores for each biomarker quantified to obtain a total score.
  • an algorithm or computer program compares the total score with a predetermined score, and uses the comparison to determine a diagnosis/prognosis.
  • one or more instructions for manually performing the above steps by a human can be provided.
  • the subject following a determination that a subject has suffers from lung squamous cell carcinoma, the subject is appropriately treated.
  • therapy is administered to treat lung squamous cell carcinoma.
  • therapy is administered to treat complications of lung squamous cell carcinoma (e.g., surgery, radiation, chemotherapy).
  • treatment comprises palliative care.
  • methods of monitoring treatment of lung squamous ceil carcinoma are provided.
  • the present methods of detecting biomarkers are carried out at a time 0.
  • the method is carried out again at a time 1 , and optionally, a time 2, and optionally, a time 3, etc., in order to monitor the progression of lung squamous cell carcinoma or to monitor the effectiveness of one or more treatments of lung squamous cell carcinoma.
  • Time points for detection may be separated by, for example at least 4 hours, at least 8 hours, at least 12 hours, at least 1 day, at least 2 days, at least 4 days, at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 6 months, or by 1 year or more.
  • a treatment regimen is altered based upon the results of monitoring (e.g., upon determining that a first treatment is ineffective).
  • the level of intervention may be altered.
  • Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.
  • the raw count RNA sequencing data for lung squamous ceil carcinoma patients were downloaded from GDC data portal.
  • the patient clinical data, including specific tumor stage and grade, are downloaded from GDC data port.
  • the SRA RNA sequencing data for normal lung tissue were downloaded from GTEx data portal through dbGaP (Table 2).
  • the two data sets were then manually curated based on the available stage and grade information from patient clinical data.
  • Genomic sequencing pipeline for RNA sequencing data Genomic sequencing pipeline for RNA sequencing data.
  • the entire RNAseq pipeline was divided into two parts for GTEx data: alignment and quantification ( Figure 1).
  • the alignment step consists of: SRA to bam conversion using SRA Toolkits (SRA Toolkit development team), bam to fastq conversion using Biobambam (Tischler G et al.,2014), and fastq to aligned bam conversion using STAR (Alex D et al,.2016).
  • the quantification step consists of: quality improvement filtering using Fixmate
  • the output from quantification step results in gene raw counts for GTEx data and is conbined with GDC gene profile for further downstream analysis.
  • the gene expression profile is then pre-filtered based on the mean expression per gene.
  • the filtered profile is then normalized using quantile metric and is converted into log2 scale.
  • Combat package (from edgeR, http://www.r-project.org ⁇ is then used to perform further normalization between GDC case, GDC control, and GTEx control to minimize the difference between normal controls from two databases ( Figure 1 ).
  • the normalized gene profile is then analyzed by linear model using R package 'limma' (http://www.r-project.org/).
  • the 50 genes with relatively low p-values and relatively large absolute value of log2 fold change were selected as our panel.
  • the selected gene expression profile was firstly normalized to z-score across all the samples.
  • Receiver-operator characteristic (ROC) analysis was conducted ( Figure 3) to evaluate the ability of the selected gene expression profile in differentiating the subjects in the testing cohort with early stage lung squamous cell carcinoma patients from those normal samples. This process was repeated 500 times using bootstrapping algorithm to get more accurate evaluation of the model. Heat map.
  • RNA sequencing data for early stage lung squamous cell carcinoma tissue and normal lung tissue were downloaded from GDC and GTEx data portal.
  • the patient clinical data, including specific tumor stage and grade, are downloaded from GDC data port.
  • the normal lung tissue data from GTEx were processed using developed RNA-seq pipeline.
  • the Random Forest based risk model stratified all subjects in training and testing cohorts into two levels of risk for progression as discussed above (normal or early stage). 50 targeted gene profiles were used as the model input. The risk scores of lung squamous cell carcinoma were calculated by the model ( Figure 2). We use 0.5 as the cutoff threshold.
  • Unsupervised hierarchical clustering analysis was applied to the targeted gene profiles to visually depict the association of the disease status with the abundance patterns of these targeted gene profiles (Figure 4). This analysis demonstrated two major clusters reflecting norma! samples and early stage lung squamous ceil carcinoma samples. The error rate of the unsupervised clustering is 0.28%, which reinforced the effectiveness of this targeted gene panel for lung squamous ceil carcinoma assessment.

Landscapes

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

Abstract

L'invention concerne des marqueurs de carcinome pulmonaire à cellules squameuses, des panels de marqueurs de carcinome pulmonaire à cellules squameuses, et des procédés d'obtention d'une représentation du taux de marqueur de carcinome pulmonaire à cellules squameuses pour un échantillon, sur la base d'un profilage d'expression de RNAseq. Ces compositions et ces procédés sont utiles dans un certain nombre d'applications, comprenant, par exemple, le diagnostic d'un carcinome pulmonaire à cellules squameuses, le pronostic d'un carcinome pulmonaire à cellules squameuses, la surveillance d'un sujet atteint d'un carcinome pulmonaire à cellules squameuses, et la détermination d'un traitement d'un carcinome pulmonaire à cellules squameuses ; en outre, l'invention concerne des systèmes, des dispositifs et des kits associés qui sont utiles dans la mise en œuvre des procédés de l'invention.
PCT/US2017/023473 2017-03-21 2017-03-21 Procédés et compositions pour la détection d'un carcinome pulmonaire à cellules squameuses à un stade précoce à l'aide d'un profilage d'expression de rnaseq Ceased WO2018174859A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US2017/023473 WO2018174859A1 (fr) 2017-03-21 2017-03-21 Procédés et compositions pour la détection d'un carcinome pulmonaire à cellules squameuses à un stade précoce à l'aide d'un profilage d'expression de rnaseq

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2017/023473 WO2018174859A1 (fr) 2017-03-21 2017-03-21 Procédés et compositions pour la détection d'un carcinome pulmonaire à cellules squameuses à un stade précoce à l'aide d'un profilage d'expression de rnaseq

Publications (1)

Publication Number Publication Date
WO2018174859A1 true WO2018174859A1 (fr) 2018-09-27

Family

ID=63586063

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2017/023473 Ceased WO2018174859A1 (fr) 2017-03-21 2017-03-21 Procédés et compositions pour la détection d'un carcinome pulmonaire à cellules squameuses à un stade précoce à l'aide d'un profilage d'expression de rnaseq

Country Status (1)

Country Link
WO (1) WO2018174859A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109852697A (zh) * 2019-03-28 2019-06-07 中国科学院上海生命科学研究院 腺鳞癌诊断的分子靶点及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006053442A1 (fr) * 2004-11-22 2006-05-26 Diagnocure Inc. Cible specifique et sensible (calml3) pour un diagnostic, un pronostic et/ou une therapie adaptee au diagnostic et au savoir-faire du domaine, d'un cancer du poumon
US20160019337A1 (en) * 2013-03-15 2016-01-21 Htg Molecular Diagnostics, Inc. Subtyping lung cancers

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006053442A1 (fr) * 2004-11-22 2006-05-26 Diagnocure Inc. Cible specifique et sensible (calml3) pour un diagnostic, un pronostic et/ou une therapie adaptee au diagnostic et au savoir-faire du domaine, d'un cancer du poumon
US20160019337A1 (en) * 2013-03-15 2016-01-21 Htg Molecular Diagnostics, Inc. Subtyping lung cancers

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHI ET AL.: "Aberrant signaling pathways in squamous cell lung carcinoma", CANCER INFORM., vol. 10, 2011, pages 273 - 85, XP055556347 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109852697A (zh) * 2019-03-28 2019-06-07 中国科学院上海生命科学研究院 腺鳞癌诊断的分子靶点及其应用

Similar Documents

Publication Publication Date Title
US10494677B2 (en) Predicting cancer outcome
AU2015249113B2 (en) Lung cancer biomarkers and uses thereof
KR101870123B1 (ko) 폐암 바이오마커 및 그것의 용도
CN103890586B (zh) 肺癌生物标记及其用途
US20120143805A1 (en) Cancer Biomarkers and Uses Thereof
WO2011031344A1 (fr) Biomarqueurs du cancer, et leurs utilisations
WO2018174861A1 (fr) Méthodes et compositions de détection du cancer du sein à un stade précoce par profilage d'expression par arn-seq
US20150160225A1 (en) Renal Cell Carcinoma Biomarkers and Uses Thereof
WO2018174860A1 (fr) Méthodes et compositions de détection d'un adénocarcinome pulmonaire à un stade précoce par profilage d'expression par arn-seq
US20160138110A1 (en) Glioma biomarkers
CN113502326B (zh) 基于生物标志物的肺动脉高压的诊断产品及其应用
WO2018174863A1 (fr) Méthodes et compositions de détection du cancer du côlon à un stade précoce par profilage d'expression par arn-seq
CN113444796B (zh) 与肺癌相关的生物标志物及其在诊断癌症中的应用
WO2018140049A1 (fr) Procédés et compositions permettant de détecter un cancer de l'ovaire à un stade précoce avec un profilage d'expression de rnaseq
WO2018174862A1 (fr) Méthodes et compositions de détection du cancer de la vessie à un stade précoce par profilage d'expression par arn-seq
US20240393337A1 (en) Lung Cancer Prediction and Uses Thereof
WO2016123058A1 (fr) Biomarqueurs pour la détection du risque de tuberculose
WO2018174859A1 (fr) Procédés et compositions pour la détection d'un carcinome pulmonaire à cellules squameuses à un stade précoce à l'aide d'un profilage d'expression de rnaseq
US20180356419A1 (en) Biomarkers for detection of tuberculosis risk
EP3736345A1 (fr) Prédicteurs génomiques du cancer de la vessie micropapillaire agressif
JP2025534223A (ja) タバコ使用状態を査定する方法
EP2607494A1 (fr) Biomarqueurs pour l'évaluation du risque de cancer des poumons
JP2025516097A (ja) 試料品質評価の方法
JP2025526256A (ja) 試料品質評価の方法
CN113322325A (zh) 基因群作为检测指标在口腔鳞癌诊断中的应用

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: 17902350

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17902350

Country of ref document: EP

Kind code of ref document: A1