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WO2018146162A1 - Biomarqueur moléculaire pour le pronostic de patients atteints de septicémie - Google Patents

Biomarqueur moléculaire pour le pronostic de patients atteints de septicémie Download PDF

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Publication number
WO2018146162A1
WO2018146162A1 PCT/EP2018/053105 EP2018053105W WO2018146162A1 WO 2018146162 A1 WO2018146162 A1 WO 2018146162A1 EP 2018053105 W EP2018053105 W EP 2018053105W WO 2018146162 A1 WO2018146162 A1 WO 2018146162A1
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sepsis
expression
expression level
bpgm
ratio
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PCT/EP2018/053105
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English (en)
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Brendon SCICLUNA
Tom Van Der Poll
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Academisch Medisch Centrum
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Publication of WO2018146162A1 publication Critical patent/WO2018146162A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates in general to the use of the expression level of particular genes and their potential to predict the mortality of sepsis. More specifically, the invention relates to the expression level of the genes BPGM and TAP2 as a prognostic biomarker for a high risk of mortality sepsis endotype.
  • the present invention is based on the following findings.
  • the poor prognosis sepsis endotype, Marsl was characterized by decreased expression in genes that function in both innate and adaptive immune mechanisms concomitant to high expression of specific cellular metabolic pathways including heme biosynthesis. Glycine accumulation, biosynthesized by serine derived from the glycolysis pathway intermediate 3-phosphoglycerate, fuels heme biosynthesis and in turn modulates ATP synthesis via oxidative phosphorylation in mitochondria(35).We therefor believe that Marsl classified patients may represent an "immunoparalyzed" endotype with poor prognosis.
  • the Mars2 and Mars4 endotypes were characterized by high expression of genes involved in proinflammatory and innate immune reactions, for example NF-kB signaling and interferon signaling, respectively.
  • Mars2 and Mars4 classified patients may represent distinct hyper-inflammatory endotypes.
  • Genes with elevated expression in the relatively lowest risk Mars3 endotype were over- represented for adaptive immune/T cell pathways.
  • Clinical trials for sepsis seeking to modify the host response have thus far yielded no beneficial effect on outcome(38).
  • a growing body argues for the re-assessment of clinical trial designs(39) to include biomarkers reflecting the status of the host response(38,40).
  • Our study shows that sepsis patients can be classified to four endotypes based on host leukocyte response signatures with distinct pathophysiologic and prognostic features.
  • endotype classification may provide more homogeneity to the notoriously heterogeneous sepsis population, can aid in identifying patient subgroups who would most likely benefit from precision therapeutics, and can be used to monitor effects of targeted therapies.
  • the invention provides a method for determining the survival prognosis of a patient suffering from sepsis, said method comprising steps of: (a) determining the expression level(s) of TAP2 and/or BPGM in a biological sample of said patient, (b) determining said expression level(s), (c) determining the risk of suffering from sepsis based on said expression level(s).
  • the invention provides a method for determining the survival prognosis of a patient suffering from sepsis, said method comprising steps of: (a) determining the expression level(s) of GADD45A and/or PCGF5 in a biological sample of said patient, (b) determining said expression level(s), (c) determining the risk of suffering from sepsis based on said expression level(s).
  • the invention provides a method for determining the survival prognosis of a patient suffering from sepsis, said method comprising steps of: (a) determining the expression level(s) of AHNAK and/or PDCD10 in a biological sample of said patient, (b) determining said expression level(s), (c) determining the risk of suffering from sepsis based on said expression level(s).
  • the invention provides a method for determining the survival prognosis of a patient suffering from sepsis, said method comprising steps of: (a) determining the expression level(s) of IFIT5 and/or GLTSCR2 in a biological sample of said patient, (b) determining said expression level(s), (c) determining the risk of suffering from sepsis based on said expression level(s).
  • said step of determining the expression level comprises the use of at least one techniques selected from the group consisting of:
  • PCR-based methods preferably RT-PCR, Quantitative RT-PCR, nucleic acid microarray analysis, isothermal DNA/RNA amplification techniques, and/or in situ hybridization, ii. immunological methods, preferably immunohistochemistry, ELISA, binding assays, and/or Western Blot, iii. and/or spectroscopical methods, preferably Raman spectroscopy, and/or Mass spectroscopy.
  • said method comprises a step of comparing said expression level(s) with a reference control or reference value.
  • said step b) comprises the step of checking whether or not the expression level of TAP2 and/or BPGM is higher or lower than a predetermined threshold level.
  • said expression levels are normalized.
  • said method comprises the step of determining the ratio of the expression levels of the of TAP2 and BPGM and determining the risk of suffering from sepsis based on said ratio.
  • the ratio of the expression levels of the of TAP2 and BPGM is compared
  • a TAP2:BPGM gene expression ratio of 1.15 or higher is associated with a poor survival prognosis.
  • said biological sample is a blood sample.
  • said patient is a human.
  • Figure 1.1 Unsupervised classification of sepsis patients and the association to clinical characteristics and outcome (discovery cohort).
  • B Silhouette width analysis illustrating stable partitioning to four molecular endotypes. Percent of patients assigned to each subtype is denoted.
  • C Gene expression heatmap illustrating the 140 gene classifier derived to categorize the patients to endotypes. Red, over-expression; turquoise, under-expression (heatmap rows). Endotypes are color coded and labeled (top bar).
  • FIG. 1.2 Assessment of sepsis molecular endotypes in the validation cohorts.
  • B-D Stratification of first validation cohort as Mars sepsis endotypes was evaluated for the association against (B) total Sequential Organ Failure Assessment (SOFA) scores, (C) septic shock, and (D) 28-day mortality by Kaplan Meier survival analysis.
  • FIG. 1.3 Biological interpretation of sepsis molecular endotypes.
  • pie charts illustrate the extent of gene expression changes with red slices denoting the number of significantly over-expressed genes (adjusted P value ⁇ 0.05 and fold expression > 1.5), blue slices denoting significant under-expression (adjusted P value ⁇ 0.05 and fold expression ⁇ -1.5) and grey slices illustrating significantly differential gene expression (adjusted P value ⁇ 0.05) but outside of the fold expression ⁇ -1.5 and > 1.5 cutoff.
  • B Correlograms illustrating the relationship of the gene expression changes in sepsis patients classified as Marsl-4 endotypes relative to healthy subjects. Rho, Spearman's correlation estimate. All correlations were significant (p ⁇ 1x10-10).
  • ROC Receiver-operator characteristics
  • AUC area-under-the-curve
  • Figure 2.1 Methodological steps employed for the unsupervised consensus clustering and gene expression classifier construction using the discovery set and subsequent validation in independent validation datasets.
  • APACHE IV Acute Physiology and Chronic Health Evaluation IV
  • Plot illustrate the Hosmer-Lemeshow test probabilities, which denote optimal calibration for a continuous net reclassification assessment (p>0.05).
  • FIG. 1 Sepsis molecular endotypes in pneumonia and abdominal sepsis
  • D, E Heatmap representation of 140 gene expression indices (rows) and patient samples stratified according to molecular endotype membership (columns) diagnosed at ICU admission with (D) pneumonia, or (E) peritonitis.
  • PRISM pediatric risk of mortality
  • FIG. 2.7 A molecular endotype model for the risk stratification of critically ill patients with sepsis.
  • the relatively high-risk Marsl sepsis endotype was defined by elevated expression of heme biosynthesis genes concomitant with pronounced under-expression of pattern recognition receptor, cytokine signaling, lymphocyte signaling and antigen presentation pathways.
  • PRR pattern recognition receptor.
  • SRS1 and 2 sepsis response signatures 1 and 2
  • X2 p chi-square test probability.
  • FIG. 2.8 Candidate sepsis endotype biomarker assessment in two validation cohorts.
  • A Dot plots of Mars2 (GADD45A:PCG F5), Mars3 (AH NAK:PDCD10) and Mars4 (I FIT5:G LTSCR2) scores in the first validation cohort from the Netherlands, and
  • B in the second validation cohort from the U K.
  • GADD45A growth arrest and DNA damage inducible alpha
  • PCGF5 polycomb group ring finger 5
  • AH NAK AH NAK nucleoprotein
  • PDCD10 programmed cell death 10
  • I FIT5 interferon induced protein with tetratricopeptide repeats 5
  • GLTSCR2 glioma tumor suppressor candidate region gene 2.
  • Horizontal black line denotes median.
  • survival prognosis refers to the prediction of the likelihood of sepsis- attributable death.
  • sepsis as used herein is defined as a Systemic Inflammatory Response Syndrome to an infective process in which severe derangement of the host immune system fails to prevent extensive 'spill over' of inflammatory mediators from a local infection focus into the systemic circulation.
  • said patient of an infection is assessed as probable or definite using Center for Disease Control and Prevention(14) and International Sepsis Forum consensus definitions(15), as described in detail (12).
  • "sepsis” is defined as the presence of infection with a probable or definite likelihood, accompanied by at least one additional parameter as described in the 2001 International Sepsis Definitions Conference (16).
  • said sepsis patient of the invention suffers from community acquired pneumonia.
  • the term "community acquired pneumonia” (sepsis) as used herein is equivalent and has been used interchangeably with the term "community acquired bacterial pneumonia.”
  • BPGM bisphosphoglycerate mutase
  • TEP2 refers to a gene encoding transporter 2, ATP binding cassette subfamily B member (RefSeq accession NC_000006.12).
  • gene biomarker refers to any or multiple of the genes of the invention.
  • biological sample refers to any sample from a patient for diagnostic, prognostic, or personalized medicinal uses and may be obtained from surgical samples, such as biopsies or fine needle aspirates, from paraffin-embedded tissues, from frozen tissue samples, from fresh tissue samples, from a fresh or frozen body fluid. Most preferably the sample contains white blood cells. However, any other suitable biological samples (e.g. bodily fluids such as blood, stool, etc..) in which the gene expression level of a gene of interest can be determined are included within the scope of the invention.
  • determining the expression level refers to the process of determining whether a gene is expressed and if this is the case assessing to which extend it is expressed. These assessments are usually carried out in parallel, but of course they can also be carried out after each other. Therefore, the process of determining gene expression may include all necessary preparatory steps know in the art such as protein, mRNA, RNA, DNA and/or cDNA preparation; measurement using techniques such as real time PCR, immunohistochemistry or microarray; basic arithmetic operations such as determining a mean value, if gene expression level for one biological sample is determined using more than one probe since the average of the probes can then be calculated in order to increase the accuracy of the inventive method; etc.
  • control refers to a biological sample or samples of a patient suffering from sepsis for determining control expression levels; and/or a predetermined expression level or ratio for each of two of biomarker expression levels and/or a predetermined cut-off level.
  • a control refers to control of BPGM and TAP2 expression levels (or in certain embodiments to expression levels of the other biomarkers genes associated with the mars 2, 3 or 4 endotypes as described herein) ; and/or a predetermined expression level or ratio for each of two of BPGM and TAP2 expression levels levels (or in certain embodiments to expression levels of the other biomarkers genes associated with the mars 2, 3 or 4 endotypes as described herein) and/or a predetermined cut-off level.
  • the control can for example be a reference profile to which test sample expression levels are compared, and/or a predetermined level or levels expressed for example as a numerical value and/or range (e.g. control range) corresponding to the biomarker levels in such sample or samples.
  • control samples with a known outcome can be used to determine a cut-off above which subjects are predicted to have an outcome (e.g. poor outcome) and below which subjects are predicted to have a different outcome (e.g. good outcome).
  • Test samples are then compared to the predetermined value determined using control samples.
  • the control can be an average, median, or calculated cut-off value (e.g. threshold) for each of 2 of BPGM and TAP2 levels (or in certain embodiments for the other biomarkers genes associated with the mars 2, 3 or 4 endotypes as described herein) and/or a composite thereof (e.g. sum) above or below which value a subject can be classified with an outcome class— e.g. good outcome or poor outcome.
  • the control is a selected value above which corresponds with an outcome and below which corresponds with another outcome.
  • a relative or normalized expression is determined to one or more internal normalization genes (e.g. internal to the test sample) which are known and/or are determined to be suitable e.g. not vary significantly due to BC and/or from patient to patient.
  • Control samples can be used to establish a fold increase relative to the normalization gene or genes. Accordingly, the control can be, for each biomarker, a ratio of the biomarker gene expression level and the level of one or more internal standardization markers in a control sample.
  • the control ratio is compared to a corresponding ratio determined for the sample. For example, if the ratio of the biomarker gene and internal standardization marker in a control sample is 1, a ratio of 1.5, 2, 2.5 or more is indicative of increased expression and a ratio of 0.8, 0.5, 0.3 or less is indicative of decreased expression.
  • the ratios can also be used to determine a cut off or threshold level or used in a SSM calculation. In such cases the control is a selected value above which is determined to predict one outcome and below which is determined to predict a different outcome.
  • the cut-off, threshold or control signature score can for example be a median level or value, or composite signature score comprising the median expression level or levels, for example the weighted expression levels, in a population of subjects.
  • a cut-off or threshold can be determined to optimize the trade-off between false negative and false positive discoveries, for example by optimizing the area under the ROC curve. It may also be desirable to define multiple thresholds, for example to assign patients to high, medium, and low risk groups.
  • the threshold(s) may be at any percentile of risk scores in the study sample, for example corresponding to the lowest 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of risk scores calculated form histologically normal margins in a population of subjects.
  • control as herein defined is distinct from for example a PCR control, no template PCR control or internal control, which is used for example with quantitative PCR.
  • an internal control is a non-biomarker gene that is expected to be expressed at relatively the same level in different samples that is used to quantify the relative amount of biomarker transcript for comparison purposes.
  • determining an expression level or "determining an expression profile” as used in reference to a biomarker means the application of a biomarker specific reagent such as a probe, primer or antibody and/or a method to a sample, for example a sample of the subject and/or a control sample, for ascertaining or measuring quantitatively, semi-quantitatively or qualitatively the amount of a biomarker or biomarkers, for example the amount of biomarker polypeptide or mRNA.
  • a biomarker specific reagent such as a probe, primer or antibody and/or a method to a sample, for example a sample of the subject and/or a control sample, for ascertaining or measuring quantitatively, semi-quantitatively or qualitatively the amount of a biomarker or biomarkers, for example the amount of biomarker polypeptide or mRNA.
  • a level of a biomarker can be determined by a number of methods including for example immunoassays including for example immunohistochemistry, ELISA, Western blot, immunoprecipitation and the like, where a biomarker detection agent such as an antibody for example, a labeled antibody, specifically binds the biomarker and permits for example relative or absolute ascertaining of the amount of polypeptide biomarker, hybridization and PCR protocols where a probe or primer or primer set are used to ascertain the amount of nucleic acid biomarker, including for example probe based and amplification based methods including for example microarray analysis, RT-PCR such as quantitative RT-PCR, serial analysis of gene expression (SAGE), Northern Blot, digital molecular barcoding technology, for example Nanostring:nCounterTM Analysis, and TaqMan quantitative PCR assays.
  • immunoassays including for example immunohistochemistry, ELISA, Western blot, immunoprecipitation and the like
  • a biomarker detection agent such as an
  • mRNA in situ hybridization in formalin-fixed, paraffin-embedded (FFPE) tissue samples or cells can be applied, such as mRNA in situ hybridization in formalin-fixed, paraffin-embedded (FFPE) tissue samples or cells.
  • FFPE paraffin-embedded
  • This technology is currently offered by the QuantiGene ® ViewRNA (Affymetrix), which uses probe sets for each mRNA that bind specifically to an amplification system to amplify the hybridization signals; these amplified signals can be visualized using a standard fluorescence microscope or imaging system.
  • This system for example can detect and measure transcript levels in heterogeneous samples; for example, if a sample has normal and tumor cells present in the same tissue section.
  • TaqMan probe-based gene expression analysis can also be used for measuring gene expression levels in tissue samples, and for example for measuring mRNA levels in FFPE samples.
  • TaqMan probe-based assays utilize a probe that hybridizes specifically to the mRNA target. This probe contains a quencher dye and a reporter dye (fluorescent molecule) attached to each end, and fluorescence is emitted only when specific hybridization to the mRNA target occurs.
  • the exonuclease activity of the polymerase enzyme causes the quencher and the reporter dyes to be detached from the probe, and fluorescence emission can occur. This fluorescence emission is recorded and signals are measured by a detection system; these signal intensities are used to calculate the abundance of a given transcript (gene expression) in a sample.
  • difference in the level refers to a measurable difference in the level or quantity of a biomarker or biomarkers associated in a test sample, compared to the control that is of sufficient magnitude to allow assessment of predicted outcome, for example a significant difference or a statistically significant difference.
  • the magnitude of the difference is sufficient for example to determine that the subject falls within a class of subjects likely to have poor survival prognosis or good survival prognosis.
  • a difference in a level of biomarker level is detected if a ratio of the level in a test sample as compared with a control is greater than 1.15 for example, a ratio of greater than 1.5, 1.7, 2, 3, 3, 5, 10, 12, 15, or more and/or a ratio less than 0.7, for example a ratio less than 0.6, 0.5, 0.4, 0.2, 0.1, 0.05 or more.
  • PCR-based methods preferably RT-PCR, Quantitative real Time PCR, nucleic acid microarray analysis, isothermal DNA/RNA amplification techniques, and/or in situ hybridization,
  • the methods of the invention involves a step of measuring the expression level of certain genes.
  • the expression level of a gene biomarker can be measured by the biomarker's mRNA level, protein level, activity level, or other quantity reflected in or derivable from the biomarker's gene or protein expression data.
  • the expression products of each of the gene biomarkers of the invention include both RNA and protein.
  • RNA products of the genes of the invention are transcriptional products of the genes of the invention and include populations of hnRNA, mRNA, and one or more spliced variants of mRNA. Protein products of the genes of the invention may also be measured.
  • the protein products of the genes of the invention include, for example, proteins, protein variants arising from spliced mRNA variants, and post translationally modified proteins.
  • any suitable means of measuring the expression of the RNA products of the genes of the invention can be used in accordance with the methods described herein.
  • the methods may utilize a variety of polynucleotides that specifically hybridize to one or more of the RNA products of the genes of the invention including, for example, oligonucleotides, cDNA, DNA, RNA, PCR products, synthetic DNA, synthetic RNA, or other combinations of naturally occurring of modified nucleotides which specifically hybridize to one or more of the RNA products of the genes of the invention.
  • Such polynucleotides may be used in combination with the methods to measure RNA expression including, for example, array hybridization, RT-PCR, nuclease protection and northern blots.
  • the expression level of the genes of the invention may be determined using array hybridization to evaluate the level of RNA expression.
  • Array hybridization utilizes nucleic acid members stably associated with a support that can hybridize with genes of the invention expression products.
  • the length of a nucleic acid member attached to the array can range from 8 to 1000 nucleotides in length and are chosen so as to be specific for the RNA products of the genes of the invention.
  • the array may comprise, for example, one or more nucleic acid members that are specific for the RNA products of the genes of the invention, or variants thereof (e.g., splice variants).
  • the nucleic acid members may be RNA or DNA, single or double stranded, and/or may be oligonucleotides or PCR fragments amplified from cDNA.
  • oligonucleotides are approximately 10-100, 10-50, 20-50, or 20-30 nucleotides in length. Portions of the expressed regions of the genes of the invention can be utilized as probes on the array. More particularly oligonucleotides complementary to the genes of the invention genes and or cDNAs derived from the genes of the invention genes are useful. For oligonucleotide based arrays, the selection of oligonucleotides corresponding to the gene of interest, which are useful as probes is well understood in the art.
  • Arrays may be constructed, custom ordered, or purchased from a commercial vendor. Various methods for constructing arrays are well known in the art.
  • the level of the expression of the RNA products of the genes of the inventions can be measured by amplifying the RNA products of the biomarkers from a sample using reverse transcription (RT) in combination with the polymerase chain reaction (PCR).
  • RT reverse transcription
  • PCR polymerase chain reaction
  • the RT can be quantitative as would be understood to a person skilled in the art.
  • Total RNA, or mRNA from a sample may be used as a template and a primer specific to the transcribed portion of a genes of the inventions is used to initiate reverse transcription.
  • Methods of reverse transcribing RNA into cDNA are well known and are described, for example, in Sambrook et al., 1989, supra.
  • Primer design can be accomplished utilizing commercially available software (e.g., Primer Designer 1.0, Scientific Software etc.) or methods that are standard and well known in the art.
  • Primer Software programs can be used to aid in the design and selection of primers include, for example, The Primer Quest software which is available through the following web site link: biotools.idtdna.com/primerquest/. Additionally, the following website links are useful when searching and updating sequence information from the Human Genome Database for use in biomarker primer design:
  • NCBI LocusLink Homepage world wide web at ncbi.nlm.nih.gov/LocusLink/
  • Ensemble Human Genome Browser world wide web at ensembl.org/Homo_sapiens, preferably using pertinent biomarker information such as Gene or Sequence Description, Accession or Sequence ID, Gene Symbol, RefSeq #, and/or UniGene #.
  • the product or amplicon length may be ⁇ 100-150 bases
  • the optimum Tm may be ⁇ 60° C, or about 58-62° C
  • the GC content may be ⁇ 50%, or about 45-55%.
  • sequences such as one or more of the following: (i) strings of three or more bases at the 3'-end of each primer that are complementary to another part of the same primer or to another primer in order to reduce primer-dimer formation, (ii) sequences within a primer that are complementary to another primer sequence, (iii) runs of 3 or more G's or C's at the 3'- end, (iv) single base repeats greater than 3 bases, (v) unbalanced distributions of G/C- and A/T rich domains, and/or (vi) a T at the 3'-end.
  • PCR provides a method for rapidly amplifying a particular nucleic acid sequence by using multiple cycles of DNA replication catalyzed by a thermostable, DNA-dependent DNA polymerase to amplify the target sequence of interest.
  • PCR requires the presence of a nucleic acid to be amplified, two single-stranded oligonucleotide primers flanking the sequence to be amplified, a DNA polymerase, deoxyribonucleoside triphosphates, a buffer and salts.
  • the method of PCR is well known in the art. PCR, is performed as described in Mullis and Faloona, 1987, Methods Enzymol., 155: 335.
  • QRT-PCR which is quantitative in nature, can also be performed to provide a quantitative measure of genes of the invention gene expression levels.
  • reverse transcription and PCR can be performed in two steps, or reverse transcription combined with PCR can be performed concurrently.
  • One of these techniques for which there are commercially available kits such as Taqman (Perkin Elmer, Foster City, Calif.), is performed with a transcript-specific antisense probe.
  • This probe is specific for the PCR product (e.g. a nucleic acid fragment derived from a gene) and is prepared with a quencher and fluorescent reporter probe complexed to the 5' end of the oligonucleotide. Different fluorescent markers are attached to different reporters, allowing for measurement of two products in one reaction.
  • Taq DNA polymerase When Taq DNA polymerase is activated, it cleaves off the quencher of the probe bound to the template by virtue of its 5'-to-3' exonuclease activity. In the absence of the quenchers, the reporters now fluoresce. The color change in the reporters is proportional to the amount of each specific product and is measured by a fluorometer; therefore, the amount of each color is measured and the PCR product is quantified.
  • the PCR reactions are performed in 96 well plates so that samples derived from many individuals are processed and measured simultaneously.
  • the Taqman system has the additional advantage of not requiring gel electrophoresis and allows for quantification when used with a standard curve.
  • a second technique useful for detecting PCR products quantitatively is to use an intercalating dye such as the commercially available QuantiTect SYBR Green PCR (Qiagen, Valencia Calif.). RT-PCR is performed using SYBR green as a fluorescent label which is incorporated into the PCR product during the PCR stage and produces a fluorescence proportional to the amount of PCR product. Additionally, other systems to quantitatively measure mRNA expression products are known including Molecular BeaconsTM. Additional techniques to quantitatively measure RNA expression include, but are not limited to, polymerase chain reaction, ligase chain reaction, Qbeta replicase (see, e.g., International Application No.
  • PCT/US87/00880 isothermal amplification method (see, e.g., Walker et al. (1992) PNAS 89:382-396), strand displacement amplification (SDA), repair chain reaction, Asymmetric Quantitative PCR (see, e.g., U.S. Publication No. US200330134307A1) and the multiplex microsphere bead assay described in Fuja et al., 2004, Journal of Biotechnology 108:193-205.
  • SDA strand displacement amplification
  • Asymmetric Quantitative PCR see, e.g., U.S. Publication No. US200330134307A1
  • the level of gene expression can be measured by amplifying RNA from a sample using transcription based amplification systems (TAS), including nucleic acid sequence amplification (NASBA) and 3SR.
  • TAS transcription based amplification systems
  • NASBA nucleic acid sequence amplification
  • 3SR 3SR
  • the nucleic acids may be prepared for amplification using conventional phenol/chloroform extraction, heat denaturation, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization.
  • the double-stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6.
  • a polymerase such as T7 or SP6.
  • the RNA's are reverse transcribed into double stranded DNA, and transcribed once with a polymerase such as T7 or SP6.
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • a labeled, nucleic acid probe is brought into contact with the amplified nucleic acid sequence of interest.
  • the probe may be conjugated to a chromophore, radiolabeled, or conjugated to a binding partner, such as an antibody or biotin, where the other member of the binding pair carries a detectable moiety.
  • detection may be carried our using Southern blotting and hybridization with a labeled probe.
  • the techniques involved in Southern blotting are well known to those of skill in the art and may be found in many standard books on molecular protocols.
  • Nuclease protection assays can be used to detect and quantitate RNA products of the genes of the inventions.
  • an antisense probe e.g., radiolabeled or nonisotopic labeled
  • hybridizes in solution to an RNA sample Following hybridization, single-stranded, unhybridized probe and RNA are degraded by nucleases.
  • An acrylamide gel is used to separate the remaining protected fragments.
  • solution hybridization can accommodate up to ⁇ 100 ⁇ g of sample RNA whereas blot hybridizations may only be able to accommodate ⁇ 20-30 ⁇ g of RNA sample.
  • RNA probes Oligonucleotides and other single-stranded DNA probes can only be used in assays containing SI nuclease.
  • the single-stranded, antisense probe must typically be completely homologous to target RNA to prevent cleavage of the probe:target hybrid by nuclease.
  • a standard Northern blot assay can also be used to ascertain an RNA transcript size, identify alternatively spliced RNA transcripts, and the relative amounts of RNA products of the genes of the inventions, in accordance with conventional Northern hybridization techniques known to those persons of ordinary skill in the art.
  • Northern blots RNA samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, crosslinked and hybridized with a labeled probe.
  • Nonisotopic or high specific activity radiolabeled probes can be used including random-primed, nick-translated, or PCR-generated DNA probes, in vitro transcribed RNA probes, and oligonucleotides.
  • sequences with only partial homology may be used as probes.
  • the labeled probe e.g., a radiolabeled cDNA, either containing the full-length, single stranded DNA or a fragment of that DNA sequence may be any length up to at least 20, at least 30, at least 50, or at least 100 consecutive nucleotides in length.
  • the probe can be labeled by any of the many different methods known to those skilled in this art.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals that fluoresce when exposed to ultraviolet light, and others. A number of fluorescent materials are known and can be utilized as labels.
  • a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • isotopes include 3H, 14C, 32P, 35S, 36CI, 51Cr, 57Co, 58Co, 59Fe, 90Y, 1251, 1311, and 186Re.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
  • the enzyme may be conjugated to the selected probe by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like.
  • bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like.
  • Any enzymes known to one of skill in the art can be utilized, including, for example, peroxidase, beta-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • U.S. Pat. Nos. 3,654,090, 3,850,752, and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
  • the expression level of a genes of the invention may also be measured by the biomarker's protein level using any art-known method.
  • Traditional methodologies for protein quantification include 2-D gel electrophoresis, mass spectrometry and antibody binding.
  • Preferred methods for assaying biomarker protein levels in a biological sample include antibody-based techniques, such as immunoblotting (western blotting), immunohistological assay, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or protein chips.
  • a biomarker-specific monoclonal antibodies can be used both as an immunoadsorbent and as an enzyme-labeled probe to detect and quantify the biomarker.
  • genes of the inventions may be immunoprecipitated from a biological sample (e.g., directly from urine or serum or from a lysate of cells, etc.) using an antibody specific for said biomarker.
  • the isolated proteins may then be run on an SDS-PAGE gel and blotted (e.g., to nitrocellulose or other suitable material) using standard procedures. The blot may then be probed with an anti- biomarker specific antibody to determine the expression level of the genes of the inventions.
  • Gel electrophoresis, immunoprecipitation and mass spectrometry may be carried out using standard techniques, for example, such as those described in Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989), Harlow and Lane, Antibodies: A Laboratory Manual (1988 Cold Spring Harbor Laboratory), G. Suizdak, Mass Spectrometry for Biotechnology (Academic Press 1996).
  • antibody As used herein, the term “antibody” (Ab) or “monoclonal antibody” (mAb) is meant to include intact molecules as well as antibody portions (such as, for example, Fab, Fab', F(ab')2, Fv, single chain Fv, or Fd) which are capable of specifically binding to a genes of the invention.
  • antibody portions such as, for example, Fab, Fab', F(ab')2, Fv, single chain Fv, or Fd
  • expression levels of genes of the inventions in a biological sample of interest are compared to the expression level of said genes in an expression level reference sample.
  • the expression level reference sample may be a biological sample derived from one or more patients determined to be suffering from sepsis.
  • the expression level reference sample serves as a standard with which to compare expression level values for each genes of the invention in a test sample.
  • An increase of the expression level of BPGM compared to the expression level values in a reference sample indicates that the patient has an increased risk of mortality from sepsis.
  • An decrease of the expression level of TAP2 compared to the expression level values in a reference sample indicates that the patient has an increased risk of mortality from sepsis.
  • BPGM gene expression was significantly higher in sepsis patients; whereas, TAP2 gene expression was significantly lower in sepsis patients.
  • genes of the invention threshold expression level values are optionally set based on one or more statistical criteria for deviation from the genes of the invention expression level values in an expression level reference sample, e.g., two or more SDs away from the value for a reference sample genes of the invention expression level.
  • the expression level reference sample is a "negative" reference sample, i.e., a sample of a healthy individual.
  • the expression level reference sample is a "positive" reference sample, i.e., a sample from a sepsis patient which has died from sepsis.
  • genes of the invention expression profiles are compared to those in both positive and negative reference samples.
  • RNA from biological samples e.g., tissues or cells
  • linear RNA amplification from single cells include, e.g., Luzzi et al. (2005), Methods Mol. Biol., 293:187-207.
  • diverse kits for high quality RNA purification are available commercially, e.g., from Qiagen (Valencia, Calif.), Invitrogen (Carlsbad, Calif.), Clontech (Palo Alto, Calif.), and Stratagene (La Jolla, Calif.).
  • the method further comprises the step of normalizing at least one of the determined gene expression levels.
  • normalization of the determined expression levels refers to the process of removing error from measured data. Normalization can be carried out against an endogenous unregulated reference gene transcript or against total cellular DNA or RNA content (molecules/g total DNA/RNA and concentrations/g total DNA/RNA). For example when using quantitative real time PCR to determine gene expression level genes, which are largely unregulated are usually assessed in parallel with the target genes. These unregulated genes are termed housekeeping genes.
  • Housekeeping gene refers to genes that usually code for proteins whose activities are essential for the maintenance of cell function. They are thus ubiquitous genes expressed in most organ, tissue and/or cell types of an organism in a mainly unregulated or only weakly regulated fashion, or regulated to a constant gene expression rate. Housekeeping genes include, without limitation, glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), Cypl, albumin, actins, e . g .
  • GPDH glyceraldehyde-3 -phosphate dehydrogenase
  • Cypl Cypl
  • albumin actins
  • HPRTl hypoxantine phsophoribosyltransferase 1
  • RPLPO large ribosomal protein
  • TFRC Transferrin receptor
  • GUS beta-glucuronidase
  • corrections and/or data processing may suitably be applied, including but not limited to background signal intensity correction, for example robust multi-average, normalization, for example using quantiles, and summarization, for example median polish and log transformation.
  • background signal intensity correction for example robust multi-average
  • normalization for example using quantiles
  • summarization for example median polish and log transformation.
  • a further preferred embodiment comprises the step of checking whether or not the expression level of said gene is higher than a predetermined threshold level.
  • threshold level refers to a level of gene expression above a certain point as determined by, for example, the receiver operator characteristic curve (employed in the derivation and validation tests herein described)or calibrator samples in a qPCR, preferably in a point-of-care qPCR test.
  • the "threshold value" is subject to the technology utilized to run the test, hence it will need to be derived afresh when the TAP2:BPGM gene expression biomarker is applied to other point-of-care testing devices.
  • the method comprises the step of comparing, arithmetically, the ratio of the expression levels of TAP2 and BPGM with a reference control or reference value and determining the risk dying from sepsis based on said ratio.
  • Using the said ratio also has the advantage of relativity thereby there is no need for normalization, for example, to a "housekeeper gene".
  • the ratio of the expression levels of TAP2 and BPGM had threshold-independent ROC AUC of 0.845 (95% CI: 0.764- 0.917).
  • a numerical threshold for the ratio of the expression levels of TAP2 and BPGM is preferably defined at 1.15.
  • probes were ranked by median absolute deviation across 306 patient samples (discovery cohort). The top 5000 ranked probes were selected and analyzed by means of the consensus clustering method 19,20 .
  • To estimate k (number of endotypes) we combined cumulative distribution functions, 19,20 silhouette width analysis 21 available in the cluster package 22 and cophenetic distance correlation analysis to evaluate clustering stability. 23
  • To construct the k endotype classifier we selected patient samples with positive silhouette widths, representing core patients per endotype.
  • Endotype biomarkers were assessed using previously described methods 5 6 .
  • Net reclassification improvement was assessed by means of a continuous model using the predictABEL method (version 1.2- 2) 26 .
  • One model encompassed only Acute Physiology and Chronic Health Evaluation (APACHE) IV scores 27 (clinical), while a second model encompassed both APACHE IV scores and sepsis endotype stratification (clinical + molecular). Unless otherwise stated, significance was demarcated at p ⁇ 0.05.
  • Sepsis molecular endotype biomarkers were derived by using previously described methods.1,2
  • the 140 gene expression indices that encompassed the endotype classifier were assessed for the best combination that classified the discovery cohort.
  • a combination was a two-gene expression ratio (score):
  • Differential gene expression analysis was firstly performed by comparing patients stratified into each of four molecular endotypes to healthy subjects, and secondly by comparing each endotype to the other endotypes. For example the latter, Marsl patient gene expression data were compared to "others", where Mars2, Mars3 and Mars4 endotypes were recoded to a single group (others). These supervised analyses were done by means of moderated t tests implemented in the limma method (version
  • Ingenuity Pathway Analysis (Ingenuity Systems IPA, www.ingenuity.com) was used to identify enrichment of genes that pertain to distinct canonical signaling pathways. The Ingenuity gene knowledgebase was selected as reference and human species specified. All other parameters were default. Significance was evaluated by Fisher's exact test adjusted p-values (adjusted p ⁇ 0.01).
  • Scicluna BP Klein Klouwenberg PM
  • van Vught LA et al. A molecular biomarker to diagnose community- acquired pneumonia on intensive care unit admission. Am J Respir Crit Care Med. 2015;192(7) :826-835.
  • glioblastoma characterized by abnormalities in PDGFRA, I DH1, EGFR, and N F1. Cancer Cell.
  • ConsensusClusterPlus a class discovery tool with confidence assessments and item tracking. Bioinformatics. 2010;26(12):1572-1573.
  • Table 2.2 Characteristics of the first validation cohort classified to four molecular endotypes
  • LR+ positive likelihood ratio.
  • LR- negative likelihood ratio.
  • BPG M bisphosphoglycerate mutase. TAP2, transporter 2, ATP binding cassette subfamily B member.
  • GADD45A growth arrest and DNA damage inducible alpha.
  • PCG F5 polycomb group ring finger 5.
  • AHNAK AH NAK nucleoprotein.
  • PDCDIO programmed cell death 10.
  • I FIT5 interferon induced protein with tetratricopeptide repeats 5.
  • GLTSCR2 glioma tumor suppressor candidate region gene 2.

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Abstract

L'invention concerne une méthode de détermination du pronostic de survie d'un patient admis dans une unité de soins intensifs (USI) ou d'un patient atteint de septicémie, comprenant des étapes de détermination du niveau d'expression d'au moins BPGM et/ou TAP2 dans un échantillon biologique dudit patient, et de comparaison dudit(desdits) niveau(x) d'expression avec un contrôle et, de détermination dudit pronostic de survie sur la base de ladite comparaison.
PCT/EP2018/053105 2017-02-07 2018-02-07 Biomarqueur moléculaire pour le pronostic de patients atteints de septicémie WO2018146162A1 (fr)

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WO2022084979A1 (fr) * 2020-10-21 2022-04-28 Indian Institute Of Science Biomarqueur pour anomalie sanguine
WO2022246553A1 (fr) 2021-05-25 2022-12-01 The University Of British Columbia Diagnostic des endotypes et/ou de la gravité d'une septicémie
WO2023014802A1 (fr) * 2021-08-06 2023-02-09 Deepull Diagnostics S.L. Systèmes et procédés de détection et de gestion de sepsis chez des patients
WO2024092190A3 (fr) * 2022-10-28 2024-06-06 Children's Hospital Medical Center Identification entraînée par apprentissage automatique de signatures d'expression génique associées à une trajectoire de dysfonctionnement d'organe multiple persistante dans une maladie critique

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FR3112210A1 (fr) * 2020-07-06 2022-01-07 bioMérieux Procédé pour déterminer le risque de survenue d’une infection associée aux soins chez un patient
WO2022008825A1 (fr) * 2020-07-06 2022-01-13 bioMérieux Procédé pour déterminer le risque de survenue d'une infection associée aux soins chez un patient
WO2022084979A1 (fr) * 2020-10-21 2022-04-28 Indian Institute Of Science Biomarqueur pour anomalie sanguine
WO2022246553A1 (fr) 2021-05-25 2022-12-01 The University Of British Columbia Diagnostic des endotypes et/ou de la gravité d'une septicémie
WO2023014802A1 (fr) * 2021-08-06 2023-02-09 Deepull Diagnostics S.L. Systèmes et procédés de détection et de gestion de sepsis chez des patients
WO2024092190A3 (fr) * 2022-10-28 2024-06-06 Children's Hospital Medical Center Identification entraînée par apprentissage automatique de signatures d'expression génique associées à une trajectoire de dysfonctionnement d'organe multiple persistante dans une maladie critique

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