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WO2019173494A1 - Compositions et procédés d'amplification de cible spécifique pour rt-pcr quantitative à cellule unique à haut débit - Google Patents

Compositions et procédés d'amplification de cible spécifique pour rt-pcr quantitative à cellule unique à haut débit Download PDF

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WO2019173494A1
WO2019173494A1 PCT/US2019/020983 US2019020983W WO2019173494A1 WO 2019173494 A1 WO2019173494 A1 WO 2019173494A1 US 2019020983 W US2019020983 W US 2019020983W WO 2019173494 A1 WO2019173494 A1 WO 2019173494A1
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panel
seq
cancer
pcr
primer
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Tim Huang
Chun-Liang Chen
Chiou-Miin WANG
Richard Hung
Wei-Ting Chao
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • aspects of the invention are in the field of medicine and diagnostics.
  • aspects are directed to primer panels for detection of cancer.
  • RT-PCR is a method of detecting mRNA expressed from a particular gene.
  • This mRNA may be used for the diagnosis of a cancer by examining the expression of cancerous genes or genes indicative of cancer.
  • the most important matter in the diagnosis of a cancer using the RT-PCR method is selection of a target gene to be detected for cancer diagnosis (i.e., a“cancer diagnosis marker”).
  • a cancer diagnosis marker is detected.
  • a cancer diagnosis marker should be expressed specifically in the cancer cell or tissue and at detectable levels in numerous cancers, but are not expressed or are differentially expressed in normal tissues.
  • Characterizing a phenotype for a subject or individual may include, but is not limited to, the diagnosis of a disease or condition or the prognosis of a disease or condition.
  • Certain embodiments are directed to methods of assessing a phenotype of cells in a biological sample comprising: isolating single cells from a biological sample from a subject; determining single cell expression of a panel of target genes using at least three primer combinations selected from CSC panel (SEQ ID NO: 1-138), SLC panel (SEQ ID NO: 139-258), GLI3 panel (SEQ ID NO:259-402), CM3.6 panel (SEQ ID NO:403-543), GBM panel (SEQ ID NO:544-83 l), PC panel (SEQ ID N0:832-900), and/or the GM panel (SEQ ID N0:90l-l044); and providing a phenotype expression profile for cells in the biological sample.
  • CSC panel SEQ ID NO: 1-138
  • SLC panel SEQ ID NO: 139-258
  • GLI3 panel SEQ ID NO:259-402
  • CM3.6 panel SEQ ID NO:403-543
  • GBM panel SEQ ID NO:544-83
  • the phenotype can be used for prognosing colon cancer, prostate cancer, glioblastoma or pancreatic cancer comprising: amplifying a target nucleic acid from single cells isolated from a biologial sample from a subject that represent a panel of target genes using primer combinations selected from CSC panel (SEQ ID NO: 1-138), SLC panel (SEQ ID NO: 139-258), GLI3 panel (SEQ ID NO:259-402), CM3.6 panel (SEQ ID NO:403- 543), GBM panel (SEQ ID NO:544-83 l), PC panel (SEQ ID N0:832-900), and/or the GM panel (SEQ ID N0:90l-l044) forming amplification products; assessing single cell expression levels of the target genes by measuring the amplification products providing a single cell expression profile.
  • the expression profile can be correlated with a particular diagnosis or prognosis.
  • primer combinations can include from 6 to about 288 primer combinations from CSC panel (SEQ ID NO: 1-138), SLC panel (SEQ ID NO: 139-258), GLI3 panel (SEQ ID NO:259-402), CM3.6 panel (SEQ ID NO:403-543), GBM panel (SEQ ID NO:544-83 l), PC panel (SEQ ID N0:832-900), and/or the GM panel (SEQ ID NO:90l- 1044).
  • kits for amplification of target nucleic acids indicative of colon cancer, prostate cancer, glioblastoma or pancreatic cancer comprising, in a compartmentalized container: a plurality of PCR primer pairs selected from primer combinations of CSC panel (SEQ ID NO: 1-138), SLC panel (SEQ ID NO: 139-258), GLI3 panel (SEQ ID NO:259-402), CM3.6 panel (SEQ ID NO:403-543), GBM panel (SEQ ID NO:544-83 l), PC panel (SEQ ID N0:832-900), and/or the GM panel (SEQ ID NO:90l- 1044) for PCR amplification of target nucleic acids.
  • CSC panel SEQ ID NO: 1-138
  • SLC panel SEQ ID NO: 139-258
  • GLI3 panel SEQ ID NO:259-402
  • CM3.6 panel SEQ ID NO:403-543
  • GBM panel SEQ ID NO:544-83 l
  • PC panel SEQ ID N0:83
  • the biological sample may comprise a single cell isolated from a biological source.
  • the biological source can be blood, a blood derivative, or a blood fraction.
  • a biological sample may be provided by removing cells from a subject, but can also be provided by using a previously isolated biological sample.
  • a tissue sample can be removed from a subject suspected of having a disease by conventional biopsy techniques.
  • a blood sample is taken from the subject.
  • the blood or tissue sample is obtained from the subject prior to initiation of radiotherapy, chemotherapy, or other therapeutic treatment.
  • isolated can refer to a cell or nucleic acid that is substantially free of other tissue material, cellular material, bacterial material, viral material, or culture medium (when produced by recombinant DNA techniques) of their source of origin.
  • A“biological source” in terms of the invention means a portion of biological tissue or biological fluid.
  • a biological source can include, but is not limited to peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood.
  • biological sources are
  • Polynucleotide refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA.
  • Polynucleotides include, without limitation single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, double- stranded, or a mixture of single- and double-stranded regions.
  • polynucleotide can also include DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature.
  • Polynucleotide also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.
  • the terms“treating” or“treatment” refer to any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the injury, pathology, or condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject's physical or mental well-being, or prolonging the length of survival.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neurological examination, and/or psychiatric evaluations.
  • Effective amount and “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a therapeutic agent to effective in achieving a particular biological or therapeutic result.
  • a therapeutically effective amount of the therapeutic agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent to elicit a desired response in the individual. Such results may include, but are not limited to, the treatment of cancer, as determined by any means suitable in the art.
  • Prognosis refers to a prediction of how a patient will progress, and whether there is a chance of recovery.
  • Cancer prognosis generally refers to a forecast or prediction of the probable course or outcome of the cancer.
  • cancer prognosis includes the forecast or prediction of any one or more of the following: duration of survival of a patient susceptible to or diagnosed with a cancer, duration of recurrence-free survival, duration of progression-free survival of a patient susceptible to or diagnosed with a cancer, response rate in a group of patients susceptible to or diagnosed with a cancer, duration of response in a patient or a group of patients susceptible to or diagnosed with a cancer, and/or likelihood of metastasis and/or cancer progression in a patient susceptible to or diagnosed with a cancer.
  • Prognosis also includes prediction of favorable responses to cancer treatments, such as a conventional cancer therapy.
  • a good or bad prognosis may, for example, be assessed in terms of patient survival, likelihood of disease recurrence, disease metastasis, or disease progression (patient survival, disease recurrence and metastasis may for example be assessed in relation to a defined time point, e.g ., at a given number of years after cancer surgery (e.g, surgery to remove one or more tumors) or after initial diagnosis).
  • a good or had prognosis may be assessed in terms of overall survival, disease- free survival or progression-free survival.
  • the expression levels or patterns of genes or associated mRNA is compared to a reference level representing the same marker.
  • the reference level may be a reference level of expression from non-cancerous tissue from the same subject.
  • reference level may be a reference level of expression from a different subject or group of subjects.
  • the reference level of expression may be an expression level obtained from tissue of a subject or group of subjects without cancer, or an expression level obtained from non-cancerous tissue of a subject or group of subjects with cancer.
  • the reference level may be a single value or may be a range of values.
  • the reference level of expression can be determined using any method known to those of ordinary skill in the art.
  • the reference level is an average level of expression determined from a cohort of subjects with cancer.
  • the reference level may also be depicted graphically as an area on a graph.
  • the reference level may comprise data obtained at the same time (e.g, in the same study) as the patient's individual data, or may be a stored value or set of values, e.g, stored on a computer, or on computer-readable media. If the latter is used, new patient data for the selected marker(s), obtained from initial or follow-up samples, can be compared to the stored data for the same marker(s) without the need for additional control experiments.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • FIG. 1 Illustration of the polymerase chain reaction (PCRTM) process.
  • FIG. 2 Comparison of Standard versus nested PCRTM primer design.
  • FIG. 3. Flowchart illustration of one embodiment of a process for primer creation and selection.
  • FIG. 4. Illustration of traditional versus nested primer results for colon cancer panels SLC and CSC.
  • FIG. 5 Illustration of specificit of GL13 and CM2.6 prostate cancer nested gene primer sets.
  • FIG. 6 Illustration of the annual incidence of colon cancer in U.S., France, Germany, Italy, Spain, and the UK.
  • FIG. 7. Illustration of the annual incidence of prostate cancer in U.S., France, Germany, Italy, Spain, and the UK.
  • FIG. 8 Illustration of the annual incidence of glioblastoma in U.S., France, Germany, Italy, Spain, and the UK.
  • FIG. 9. Illustration of the annual incidence of pancreatic cancer in U.S., France,
  • compositions and methods described herein can be used to develop or conduct diagnostic and/or prognostic assays that target specific types of cancer.
  • Particular aspects include assays that address colon cancer, prostate cancer, glioblastoma, or pancreatic cancer.
  • Most cancers are diagnosed and staged by symptomatic, physical, imaging and, possibly, blood examinations.
  • current genetic characterizations are done after initial diagnosis and staging to determine treatment options or prognosis.
  • the genetic markers currently assessed for each type of cancer are listed below (Table 1). Some of these assessments are expression level testing of the gene but most are simply detecting the presence of a particular transcriptional alteration for that gene. Table 1.
  • compositions and methods described herein can be apply to four identified types of cancer: Colorectal Cancer, Prostate Cancer, Glioblastoma, and Pancreatic Cancer.
  • Colorectal Cancer Prostate Cancer
  • Glioblastoma Glioblastoma
  • Pancreatic Cancer As a diagnostic/prognostic tool, the number of new cases diagnosed annually drive the need for such compositions and/or methods.
  • the annual incidence of these types of cancer in the US and the five major European markets (France, Germany, Italy, Spain and UK) is summarized in the table below (Table 2).
  • PCR primers have been designed that allow qRT-PCR to be run for multiple gene expression products simultaneously.
  • the PCR primers are designed to allow a single laboratory procedure to be run on a single cell to quantify the expression of multiple genes.
  • the process was developed for single cell SYBR green (dye for detection) analysis using the Fluidigm BioMark HD system, but can be adapted to other systems as well.
  • the primers described herein can be used in Polymerase Chain Reaction (PCR) laboratory tests.
  • the PCR tests can detect the presence of specific fragments of nucleic acids.
  • Quantitative PCR qPCR is a variation that is able to return a quantitative measurement of the amount of the specific nucleic acid in the sample.
  • RT-PCR or qRT-PCR Reverse Transcriptase PCR
  • qRT-PCR is often used to quantify how actively a gene is being expressed (by transcription into mRNA) in the cell.
  • PCR works by using primer nucleic acid sequences that are complimentary (bind to) the ends of the nucleic acid fragment being targeted. These primers bind and provide the double stranded area in the molecule needed for the reverse transcriptase enzyme to duplicate the sequence between the two primer fragments. The result of this process is a complete nucleic acid fragment. This process is repeated numerous times (called cycles) to amplify the target nucleic acid so it is detectable.
  • PCR can be run with high sensitivity on extremely small samples, possibly even single cells. However, typically only one nucleic acid fragment can be targeted at a time. Targeting more than one can cause primers to anneal to each other non-specifically instead of the target sequence. This can result in erroneous results - false amplification and false positives.
  • Standard RT-PCR uses one set of primers per gene for both the pre-amplification and amplification phases. To reduce the specificity of the test, the research community has begun to use nested primers for each gene: one pair of outer primers for pre-amplification and one inner primer that is in either forward or reverse orientation used with one of the outer primers for the amplification phase.
  • the inventors have devised a methodology to select multiple sets of nested primers that do not interact with each other or interact in a insignificant way. These primers can be used in a single PCR run to detect several target nucleic acids at the same time. The ability to detect an entire panel of target gene products at the same time allows in depth testing to be performed on extremely small samples (single cells).
  • the primers were designed using web-based software, Primer Blast (available on the world wide web at ncbi.nlm.nih.gov/tools/primer-blast/), with a high stringency of selective parameters.
  • the resulting primers were then subjected to primer dimer check using PriDimerCheck software (available at URL biocompute.bmi.ac.cn/MPprimer/primer_dimer.html).
  • Primers with a high guanine (G) content tend to bind more specifically whereas those with a low G content bind less discriminately. Therefore, the inventors selected ones with a G > 5 and redesigned those with a lower G until they met the criteria.
  • Panels of primers have been developed to assess subjects for colon cancer, prostate cancer, glioblastoma, or pancreatic cancer.
  • Primers and methods described herein can be used in diagnostic/prognostic assays targeting a specific type of cancer. Genetic characterizations of cancers are usually done after initial diagnosis and staging to determine either treatment options or prognosis. Some of these assessments are expression level testing, but most are simply detecting the presence of a particular genetic mutation. Currently, the presence of a genetic mutation is identified by PCR or FISH (fluorescent in-situ hybridization). The expression level of a particular gene is usually determined by qPCR.
  • Embodiments described herein have at least the following advantages over the current standard of care: (a) Ability to target multiple gene products with a single cell-sized sample; (b) high throughput, faster and less expensive testing; and (c) Improved specificity which could lead to improved correlations between tumor genetic markers and disease prognosis.
  • SLC Primer Panel - A 120 memberset of nested RT-PCR primers targeting the solute carrier gene expression in Colon Cancer.
  • GLI3 Primer Panel - A 144 mRNA set of nested RT-PCR primers targeting the GLI3 activated genes in Prostate Cancer.
  • CM3.6 Primer Panel - A 140 member set of nested RT-PCR primers targeting the metastasis expression of Prostate Cancer.
  • GBM Primer Panel - A 288 member set of nested RT-PCR primers targeting Glioblastoma (Brain Cancer).
  • BC Primer Panel - A 144 mRNA set of nested RT-PCR primers targeting the metabolites of circulating tumor cells (Breast Cancer).
  • a biomarker is an organic biomolecule that is differentially present in a sample taken from a subject of one phenotypic status (e.g ., having a disease) as compared with another phenotypic status (e.g., not having the disease).
  • a biomarker is differentially present between different phenotypic statuses if the mean or median expression level of the biomarker in the different groups is calculated to be statistically significant. Common tests for statistical significance include, among others, t-test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney and odds ratio.
  • Biomarkers, alone or in combination provide measures of relative risk that a subject belongs to one phenotypic status or another. As such, they are useful as markers for disease (diagnostics), therapeutic effectiveness of a drug (theranostics) and of drug toxicity.
  • aspects of the current invention seek to develop a method for identifying patients at the risk for cancers based on gene expression profiling using CSC, SLC, GLI3, CM3.6, GBM, PC, or GM primer panels for amplifying nucleic acids to provide a biomarker panel for assessment of single cells.
  • the biomarkers of this invention can be measured or detected by using PCR. Accordingly, in the first aspect of the present invention, a method is provided for determining gene expression in a sample, and in particular a single cells form a sample. Generally, the method includes at least the following steps: (1) obtaining a sample from a patient; (2) determining the expression of a panel of genes in single cells in the sample; and (3) assess the gene expression profiles obtained. [0052] In some embodiments, a plurality of test genes are amplified using the CSC, SLC,
  • the primer panel can include primers selected from SEQ ID NO: l to 138, or SEQ ID NO: 139 to 258, or SEQ ID NO:259-402, or SEQ ID NO:403-543, or SEQ ID NO:544-83 l, or SEQ ID N0:832-900, or SEQ ID N0:90l-l044.
  • the sample used in the method may be a sample derived from prostate, colon, breast, brain or neurolic tissue, e.g., by way of biopsy or surgery.
  • the sample may also be cells naturally shedded by prostate, colon, brain or neurolic tissue, e.g., into blood, urine, sputum, etc.
  • Samples from an individual diagnosed of cancer may be used for cancer prognosis in accordance with the present invention.
  • analytical methods may be performed on a tumor sample from a patient identified as having cancer. Such a method includes at least the following steps: (1) obtaining a sample or tumor sample from a patient identified as having cancer; (2) determining the expression of a panel of genes in a cell or cells from the sample or tumor sample using the primer panels described herein; and (3) providing an assessment of the singel cell(s) gene expression profile(s).
  • the method also may be performed on a sample from a patient who has not been diagnosed as having cancer, but may be suspected of having cancer.
  • the sample may be a tissue biopsy or surgical sample directly from the organ (e.g., prostate, colon, neurologic, or brain), or cells in a bodily fluid (e.g, blood or urine) shed from such an organ.
  • the sample is from prostate or a subject having or suspected of having prostate cancer, and the primer panel includes primers having a sequence selected from SEQ ID N0:832-900.
  • the amount of RNA transcribed from the panel of genes including test genes is measured in cells present in the sample.
  • the amount of RNA of one or more housekeeping genes in cell from the sample can also be measured, and used to normalize or calibrate the expression of the test genes.
  • sample means any biological sample from a patient (including apparently healthy patients). Examples include fluids (e.g ., blood, urine, sputum, pleural fluid, semen, saliva, etc.), tissues (e.g., skin, bone, soft tissue from any particular organ or organ system, etc.), waste products (e.g, stool), etc.
  • samples includes any material extracted, purified, amplified, or derived from the preceding.
  • tissue sample means any“sample” containing one or more tumor cells, or one or more tumor-derived RNA, and obtained from a patient or subject.
  • a tissue sample obtained from a tumor tissue of a cancer patient is a useful tumor sample in the present invention.
  • the tissue sample can be an FFPE sample, or fresh frozen sample, and in some embodiments contain largely tumor cells.
  • a single malignant cell from a cancer patient's tumor is also a useful tumor sample.
  • Such a malignant cell can be obtained directly from the patient's tumor, or purified from the patient's bodily fluid such as blood and urine.
  • a bodily fluid such as blood, urine, sputum and saliva containing one or tumor cells, or tumor-derived RNA or proteins, can also be useful as a tumor sample for purposes of practicing the present invention.
  • normalizing genes referred to the genes whose expression is used to calibrate or normalize the measured expression of the gene of interest (e.g, test genes).
  • the expression of normalizing genes should be independent of cancer outcome/prognosis, and the expression of the normalizing genes is very similar among all cells. The normalization ensures accurate comparison of expression of a test gene between different cells.
  • housekeeping genes known in the art can be used.
  • Housekeeping genes are well known in the art, with examples including, but are not limited to, GUSB (glucuronidase, beta), HMBS (hydroxymethylbilane synthase), SDHA (succinate dehydrogenase complex, subunit A, flavoprotein), UBC (ubiquitin C) and YWHAZ (tyrosine 3 -monooxygenase/tryptophan 5 -monooxygenase activation protein, zeta polypeptide).
  • GUSB glucose curonidase, beta
  • HMBS hydroxymethylbilane synthase
  • SDHA succinate dehydrogenase complex, subunit A, flavoprotein
  • UBC ubiquitin C
  • YWHAZ tyrosine 3 -monooxygenase/tryptophan 5 -monooxygenase activation protein, zeta polypeptide.
  • GUSB glucose curonidase, beta
  • HMBS hydroxymethylbilane synthas
  • RNA levels for the genes In the case of measuring RNA levels for the genes, one convenient and sensitive approach is real-time quantitative PCR (qPCR) assay, following a reverse transcription reaction.
  • qPCR quantitative PCR
  • a cycle threshold C t is determined for each test gene and each normalizing gene, i.e., the number of cycle at which the fluorescence from a qPCR reaction above background is detectable.
  • the diagnosis method is used for diagnosing prostate cancer, colon cancer, pancreatic cancer, or brain cancer, in biopsy or surgical samples, or in cells from prostate, colon, pancreaas, or brain in a bodily fluid such as blood or urine.
  • the sample is a sample for which a diagnosis is ambiguous ( e.g ., not clear whether it is cancerous).
  • the sample is a sample from a subject that upon pathological or other preliminary analysis indicated a diagnosis of no cancer, for which the methods, kits, systems, etc. of the present invention may be used to either confirm the diagnosis of no cancer or to indicate the patient has cancer or has an increased likelihood of cancer.
  • the sample is a bodily fluid or waste sample for which the methods, kits, systems, etc. of the present invention may be as a screen to indicate the patient (e.g., apparently healthy patient, patient suspected of having cancer, patient at increased risk of cancer) has cancer or has an increased likelihood of cancer.
  • kits for the detection or assessment of cancer(s) that include one or more primer set as described herein.
  • the present invention further provides a diagnostic kit useful in the above methods, the kit generally comprising, in a compartmentalized container, a plurality of oligonucleotides having a nucleotide sequence of of one or more of SEQ ID NO: l to 138, or SEQ ID NO: 139 to 258, or SEQ ID NO:259-402, or SEQ ID NO:403-543, or SEQ ID NO:544-83 l, or SEQ ID N0:832-900, or SEQ ID N0:90l-l0440.
  • the invention provides a diagnostic kit for diagnosing cancer in a patient comprising the above components and reagents needed to use the primers to amplify target nucleic acids.
  • the invention provides a diagnostic kit for prognosing cancer in a patient diagnosed of prostate cancer, colon cancer, pancreatic cancer or brain cancer, comprising the above components.
  • the oligonucleotides can be primers suitable for PCR amplification of the target nucleic acids.
  • the kit consists essentially of, in a compartmentalized container, a first plurality of PCR reaction mixtures for PCR amplification, and wherein each reaction mixture comprises a PCR primer pair for PCR amplifying one of the target nucleic acids.
  • kits for practicing the prognosis of the present invention.
  • the kit may include a carrier for the various components of the kit.
  • the carrier can be a container or support, in the form of, e.g., bag, box, tube, rack, and is optionally compartmentalized.
  • the carrier may define an enclosed confinement for safety purposes during shipment and storage.
  • the kit includes various components useful in determining the status of one or more nucleic acid and optionally one or more housekeeping gene markers, using the above-discussed methods and compositions.
  • the kit many include oligonucleotides of Table 3, 4, 5, 6, 7, 8, and/or 9. Such oligonucleotides can be used as PCR primers in RT-PCR reactions.
  • the kit comprises reagents (e.g., probes or primers) for determining the expression level of a panel of nucleic acids.
  • the oligonucleotides in the detection kit can be labeled with any suitable detection marker including but not limited to, radioactive isotopes, fluorephores, biotin, enzymes (e.g., alkaline phosphatase), enzyme substrates, ligands and antibodies, etc. See Jablonski et ah, Nucleic Acids Res., 14:6115-6128 (1986); Nguyen et ah, Biotechniques, 13: 116-123 (1992); Rigby et ah, J. Mol. Biol., 113:237-251 (1977).
  • the oligonucleotides included in the kit are not labeled, and instead, one or more markers are provided in the kit so that users may label the oligonucleotides at the time of use.
  • the detection kit of this invention includes, but are not limited to, DNA polymerase, reverse transcriptase, deoxyribonucleotides, other primers suitable for the amplification of a target DNA sequence, and the like.
  • the detection kit in some embodiments includes instructions on using the kit for practice the prognosis method of the present invention using human samples. VI. Examples
  • the inventors have developed a set of methods for primer design and designed various panels of nested primers for genes involved in progression in cancer stem cells of colon cancer and prostate cancer, as well as pancreatic cancer and brain cancer.
  • the nested primers are composed of three primers, one pair of outer primers and one inner primer that is in either forward or reverse orientation.
  • the gene panels are SLC, CSC for colon cancer as well as GLI3 and CM3.6 for prostate cancer, GBM for brain cancer, BM for breast cancer, and PC for pancreatic cancer.
  • the primers were designed using web- based Primer Blast (See world wide web at URL ncbi.nlm.nih.gov/tools/primer-blast/) with high stringency of selective parameters. The primers were then subject to primer dimer check with PriDimerCheck software (available at URL biocompute.bmi.ac.cn/MPprimer/primer_dimer.html). Those primers generate potential primer dimer with AG >-5 are selected and those primers with primer dimer showing lower AG are prone to the formation of nonspecific primer dimer amplification and redesigned until they meet the set criteria.
  • the gene panels were generated according to the set standards and tested for gene expression evaluation and specificity test.
  • the specificity of primers of SLC and CSC in single cell qRT-PCR was tested on colon cancer cells.
  • the expression of 48 genes was measured with nested primer sets as compared the traditional non-nested method.
  • the primer dimers and nonspecific amplification were present in 482 (20.9%) and 292 (15.3%) qRT- PCR in two independent set of single colon cancer cells using non-nested method, whereas nested primer method yielded high quality of specific amplification with only 13 (0.56%) and 10 (0.46%) qRT-PCR generating nonspecific amplification.
  • the SLC and CSC nested primer panels were tested against traditional PCR on two types of HT29 colon cancer cells.
  • the traditional PCR resulted in primer dimers and non-specific amplification in 15-20% of the runs.
  • the nested primers result in errant effects in less than 1% of the tests.

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Abstract

Certains modes de réalisation concernent des procédés et des compositions pour caractériser un phénotype cellulaire par analyse de cellules uniques, en particulier de cellules uniques présentes dans un échantillon biologique dérivé d'un sujet. La caractérisation d'un phénotype pour un sujet ou un individu peut comprendre, mais sans s'y limiter, le diagnostic d'une maladie ou d'une affection ou le pronostic d'une maladie ou d'un affection.
PCT/US2019/020983 2018-03-06 2019-03-06 Compositions et procédés d'amplification de cible spécifique pour rt-pcr quantitative à cellule unique à haut débit Ceased WO2019173494A1 (fr)

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