WO2022186821A1 - Méthodes et compositions pour fournir une évaluation de prééclampsie à l'aide de leptine et de céramide - Google Patents

Méthodes et compositions pour fournir une évaluation de prééclampsie à l'aide de leptine et de céramide Download PDF

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Publication number
WO2022186821A1
WO2022186821A1 PCT/US2021/020417 US2021020417W WO2022186821A1 WO 2022186821 A1 WO2022186821 A1 WO 2022186821A1 US 2021020417 W US2021020417 W US 2021020417W WO 2022186821 A1 WO2022186821 A1 WO 2022186821A1
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WIPO (PCT)
Prior art keywords
preeclampsia
markers
ceamide
lep
panel
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PCT/US2021/020417
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English (en)
Inventor
Bruce Xuefeng Ling
Limin Chen
Harvey J. COHEN
Shiying Hao
Doff B. Mcelhinney
Karl G. SYLVESTER
Lu Tian
Ronald J. WONG
Gary M. SHAW
David K. Stevenson
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Mprobe Inc.,
The Board Of Trustees Of The Leland Stanford Junior
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Application filed by Mprobe Inc.,, The Board Of Trustees Of The Leland Stanford Junior filed Critical Mprobe Inc.,
Priority to PCT/US2021/020417 priority Critical patent/WO2022186821A1/fr
Publication of WO2022186821A1 publication Critical patent/WO2022186821A1/fr

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    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour

Definitions

  • the panel comprises one or more preeclampsia markers selected from the group consisting of Leptin (LEP), Ceramide (d18: 1/25:0), Ceramide (d 18: 1/26:0).
  • the method further comprises providing a report of the preeclampsia marker level representation.
  • the preeclampsia marker representation is a preeclampsia score.
  • the method further comprises comparing the preeclampsia marker level representation to a preeclampsia phenotype determination element, and providing a preeclampsia diagnosis for the subject based on the comparison.
  • the subject has symptoms of preeclampsia.
  • the subject is asymptomatic for preeclampsia.
  • the subject has one or more risk factors associated with preeclampsia.
  • the subject has no risk factors associated with preeclampsia.
  • the methods may be particularly suitable for certain pregnant women, such as those that have history of preeclampsia, have obesity, have babies less than two years or more than 10 years apart, are older than 40, have history of certain conditions including chronic high blood pressure, migraine headaches, type 1 or type 2 diabetes, kidney disease, a tendency to develop blood clots, or lupus.
  • the woman can be subject to a procedure that helps ameliorate the preeclampsia.
  • procedures include, without limitation, medications to lower blood pressure, use of corticosteroids, anticonvulsant medication such as magnesium sulfate, bed rest, and consideration of delivery if the diagnosis was made at or after 37 gestational weeks.
  • Figure 1 Study outline of discovery and testing of PE biomarkers.
  • FIG. Serial blood sampling from each normal term and PE subject at different GAs. Times of sample collections, infant deliveries, and confirmatory PE diagnoses of individual women (denoted by each row) are represented by black circles, black squares, and red-filled triangles, respectively.
  • FIG. 1 Concentrations in maternal serum of LEP, Ceramide (d 18: 1/25:0), and Ceramide (d 18: 1/26:0) as a function of gestational age at blood draw in the discovery cohort. Loess smooth lines were plotted for PE and controls, respectively.
  • FIG. 1 Concentration ratio of LEP/Ceramide (d 18: 1 /25:0) (Left) and LEP/Ceramide (d18:1/25:0) (Right) in maternal serum as a function of gestational age at blood draw in the discovery cohort. Loess smooth lines were plotted for PE and controls, respectively.
  • FIG. 1 Concentration ratio of LEP/Ceramide (d18:1/25:0) (Left) and LEP/Ceramide (d18:1/25:0) (Right) in maternal serum as a function of gestational age at blood draw in the discovery cohort. LEP was normalized by BMI prior to pregnancy. Loess smooth lines were plotted for PE and controls, respectively. Bottom: ROCAUC in different GA windows of LEP/Ceramide (d18:1/25:0) ratio, LEP/Ceramide (d18:1/26:0) ratio, and sFlt-1/PIGF in differentiating impending PE from normal. 25-0 Cer: Ceramide (d18:1/25:0). 25-0 Cer: Ceramide (d18: 1/26:0).
  • FIG. 7 Time-to-event analysis of the LEP/Ceramide (d 18: 1 /25:0) ratio and sFlt-1/PIGF ratio in the testing cohort. LEP was normalized by BMI prior to pregnancy. X axis represents the time to confirmative diagnosis of PE, and Y axis represents % of patients identified by the marker as having impending PE.
  • Preeclampsia markers, preeclampsia marker panels, and methods for obtaining a preeclampsia marker level representation for a sample are provided. These compositions and methods find use in a number of applications, including, for example, diagnosing preeclampsia, prognosing a preeclampsia, monitoring a subject with preeclampsia, and determining a treatment for preeclampsia. In addition, systems, devices and kits thereof that find use in practicing the subject methods are provided.
  • aspects of the subject invention include methods, compositions, systems and kits that find use in providing a preeclampsia assessment, e.g. diagnosing, prognosing, monitoring, and/or treating preeclampsia in a subject.
  • preeclampsia or “pre-eclampsia” it is meant a multisystem complication of pregnancy that may be accompanied by one or more of high blood pressure, proteinuria, swelling of the hands and face/eyes (edema), sudden weight gain, higher-than-normai liver enzymes, and thrombocytopenia.
  • Preeclampsia typically occurs in the third trimester of pregnancy, but in severe cases, the disorder occurs in the second trimester, e.g., after about the 22 nd week of pregnancy. If unaddressed, preeclampsia can lead to eclampsia, i.e. seizures that are not related to a preexisting brain condition.
  • diagnosis a preeclampsia or "providing a preeclampsia diagnosis,” it is generally meant providing a preeclampsia determination, e.g. a determination as to whether a subject (e.g.
  • a subject that has clinical symptoms of preeclampsia, a subject that is asymptomatic for preeclampsia but has risk factors associated with preeclampsia, a subject that is asymptomatic for preeclampsia and has no risk factors associated with preeclampsia) is presently affected by preeclampsia; a classification of the subject’s preeclampsia into a subtype of the disease or disorder; a determination of the severity of preeclampsia; and the like.
  • a preeclampsia or “providing a preeclampsia prognosis,” it is generally meant providing a preeclampsia prediction, e.g. a prediction of a subject's susceptibility, or risk, of developing preeclampsia; a prediction of the course of disease progression and/or disease outcome, e.g.
  • monitoring it is generally meant monitoring a subject's condition, e.g. to inform a preeclampsia diagnosis, to inform a preeclampsia prognosis, to provide information as to the effect or efficacy of a preeclampsia treatment, and the like.
  • treating a preeciampsia it is meant prescribing or providing any treatment of a preeclampsia in a mammal, and indudes: (a) preventing the preeclampsia from occurring in a subject which may be predisposed to preeclampsia but has not yet been diagnosed as having it; (b) inhibiting the preeclampsia, i.e., arresting its development; or (c) relieving the preeclampsia, i.e., causing regression of the preeclampsia. [0025] In describing the subject invention, compositions useful for providing a preeclampsia assessment will be described first, followed by methods, systems and kits for their use.
  • the level(s) of preeclampsia markers) in the biological sample from an individual are evaluated.
  • the level of one or more preeclampsia markers in the subject sample may be evaluated by any convenient method.
  • preeclampsia gene expression levels may be detected by measuring the levels/amounts of one or more nucleic acid transcripts, e.g. mRNAs, of one or more preeclampsia genes.
  • Protein markers may be detected by measuring the levels/amounts of one or more proteins/polypeptides.
  • any convenient protocol for evaluating protein levels may be employed wherein the level of one or more proteins in the assayed sample is determined.
  • one representative and convenient type of protocol for assaying protein levels is ELISA.
  • ELISA and ELISA-based assays one or more antibodies specific for the proteins of interest may be immobilized onto a selected solid surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate.
  • the assay plate wells are coated with a non-specific "blocking" protein that is known to be antigenically neutral with regard to the test sample such as bovine serum albumin (BSA), casein or solutions of powdered milk.
  • BSA bovine serum albumin
  • the immobilizing surface is contacted with the sample to be tested under conditions that are conducive to immune complex (antigen/antibody) formation.
  • Such conditions include diluting the sample with diluents such as BSA or bovine gamma globulin (BGG) in phosphate buffered saline (PBS)ZTweenor PBSATriton-X 100, which also tend to assist in the reduction of nonspecific background, and allowing the sample to incubate for about 2-4 hrs at temperatures on the order of about 25°-27’C (although other temperatures may be used).
  • PBS phosphate buffered saline
  • PBSATriton-X 100 phosphate buffered saline
  • An exemplary washing procedure includes washing with a solution such as PBS/Tween, PBS/Triton-X 100, or borate buffer.
  • the occurrence and amount of immunocomplex formation may then be determined by subjecting the bound immunocomplexes to a second antibody having specificity for the target that differs from the first antibody and detecting binding of the second antibody.
  • the second antibody will have an associated enzyme, e.g. urease, peroxidase, or alkaline phosphatase, which will generate a color precipitate upon incubating with an appropriate chromogenic subskate.
  • the amount of label is quantified, for example by incubation with a chromogenic substrate such as urea and bromocresol purple in the case of a urease label or 2,2'-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of a peroxidase label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.
  • a chromogenic substrate such as urea and bromocresol purple in the case of a urease label or 2,2'-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of a peroxidase label.
  • Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.
  • the preceding format may be altered by first binding the sample to the assay plate. Then, primary antibody is incubated with the assay plate, followed by detecting of bound primary antibody using a labeled second antibody with spedficity for the primary antibody.
  • the solid substrate upon which the antibody or antibodies are immobilized can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin mixture particles, etc.
  • the substrate may be chosen to maximize signal to noise ratios, to minimize background binding, as well as for ease of separation and cost. Washes may be effected in a manner most appropriate for the substrate being used, for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, or rinsing a bead, pupe, chromatograpic column or filter with a wash solution or solvent.
  • non-ELISA based-methods for measuring the levels of one or more proteins in a sample may be employed.
  • Representative examples indude but are not limited to mass spectrometry, proteomic arrays, xMAPTM microsphere technology, flow cytometry, western blotting, and immunohistochemistry.
  • the level of at least one preeclampsia marker may be evaluated by deteding in a patient sample the amount or level of one or more RNA transcripts or a fragment thereof encoded by the gene of interest to arrive at a nucleic acid marker representation.
  • the level of nucleic acids in the sample may be deteded using any convenient protocol. While a variety of different manners of deteding nucleic adds are known, such as those employed in the field of differential gene expression analysis, one representative and convenient type of protocol for generating marker representations is array-based gene expression profiling protocols.
  • Such applications are hybridization assays in which a nucleic acid that displays "probe" nudeic acids for each of the genes to be assayed/profiled in the marker representation to be generated is employed.
  • a sample of target nudeic acids is first prepared from the initial nucleic add sample being assayed, where preparation may include labeling of the target nudeic adds with a label, e.g., a member of signal produdng system.
  • the sample is contacted with the array under hybridization conditions, whereby complexes are formed between target nucleic adds that are complementary to probe sequences attached to the array surface. The presence of hybridized complexes is then detected, either qualitatively or quantitatively.
  • the resultant data provides information regarding levels in the sample for each of the markers that have been probed, wherein the information is in terms of whether or not the marker is present and, typically, at what level, and wherein the data may be both qualitative and quantitative.
  • the methods provide a reading or evaluation, e.g., assessment, of whether or not the target marker, e.g., nucleic add or protein, is present in the sample being assayed.
  • the methods provide a quantitative detection of whether the target marker is present in the sample being assayed, i.e., an evaluation or assessment of the actual amount or relative abundance of the target analyte, e.g., nucleic acid or protein in the sample being assayed.
  • the quantitative detection may be absolute or, if the method is a method of detecting two or more different analytes, e.g., target nucleic acids or protein, in a sample, relative.
  • the term "quantifying" when used in the context of quantifying a target analyte, e.g., nucleic acid(s) or protein(s), in a sample can refer to absolute or to relative quantification.
  • Absolute quantification may be accomplished by inclusion of known concentrations ) of one or more control analytes and referencing the detected level of the target analyte with the known control analytes (e.g., through generation of a standard curve).
  • relative quantification can be accomplished by comparison of detected levels or amounts between two or more different target analytes to provide a relative quantification of each of the two or more different analytes, e.g., relative to each other.
  • the measurements of the one or more preeclampsia markers may be analyzed individually to develop a preeclampsia profile.
  • a “preeclampsia profile” is the normalized level of one or more preeclampsia markers in a patient sample, for example, the normalized level of serological protein concentrations in a patient sample.
  • a profile may be generated by any of a number of methods known in the art. For example, the level of each marker may be log 2 transformed and normalized relative to the expression of a selected housekeeping gene, or relative to the signal across a whole panel, etc. Other methods of calculating a preeclampsia profile will be readily known to the ordinarily skilled artisan.
  • a preeclampsia score for a patient sample may be calculated by any of a number of methods and algorithms known in the art for calculating biomarker scores. For example, weighted marker levels, e.g. log 2 transformed and normalized marker levels that have been weighted by, e.g., multiplying each normalized marker level to a weighting factor, may be totaled and in some cases averaged to arrive at a single value representative of the panel of preeclampsia markers analyzed.
  • weighted marker levels e.g. log 2 transformed and normalized marker levels that have been weighted by, e.g., multiplying each normalized marker level to a weighting factor
  • the weights may be reflective of the importance of each marker to the specifidty, sensitivity and/or accuracy of the marker panel in making the diagnostic, prognostic, or monitoring assessment. Such weights may be determined by any convenient statistical machine learning methodology, e.g. Principle Component Analysis (PCA), linear regression, support vector machines (SVMs), and/or random forests of the dataset from which the sample was obtained may be used.
  • PCA Principle Component Analysis
  • SVMs support vector machines
  • weights for each marker are defined by the dataset from which the patient sample was obtained.
  • weights for each marker may be defined based on a reference dataset, or "training dataset”.
  • the expression, e.g. polypeptide level, of only one marker is evaluated to produce a marker level representation.
  • the levels of two or more, i.e. a panel, markers is evaluated. Accordingly, in the subject methods, the expression of at least one marker in a sample is evaluated.
  • the evaluation that is made may be viewed as an evaluation of the proteome, as that term is employed in the art.
  • the subject methods of determining or obtaining a preeclampsia marker representation for a subject further comprise providing the preeclampsia marker representation as a report.
  • the subject methods may further indude a step of generating or outputting a report providing the results of a preeclampsia marker evaluation in the sample, which report can be provided in the form of an electronic medium (e.g., an electronic display on a computer monitor), or in the form of a tangible medium (e.g., a report printed on paper or other tangible medium). Any form of report may be provided, e.g. as known in the art or as described in greater detail below.
  • the marker level representation may be employed to diagnose a preeclampsia; that is, to provide a determination as to whether a subject is affected by preeclampsia, the type of preeclampsia, the severity of preeclampsia, etc.
  • the subject may present with dinical symptoms of preeclampsia, e.g. elevated blood pressure (e.g. 140/90 mm/Hg or higher), proteinuria, sudden weight gain (over 1 -2 days or more than 2 pounds a week), water retention (edema), elevated liver enzymes, and/or thrombocytopenia (a depressed platelet count less than 100,000).
  • subject may be asymptomatic for preeclampsia but has risk factors associated with preeclampsia, e.g. a medical condition such as gestational diabetes, type I diabetes, obesity, chronic hypertension, renal disease, a thrombophilia; African-American or NHL descent; age of greater than 35 years or less than 20 years; a family history of preeclampsia; nulliparity; preeclampsia in a previous pregnancy; and/or stress.
  • the subject may be asymptomatic for preeclampsia and have no risk factors associated with preeclampsia.
  • the preeclampsia marker level representation may be employed to prognose a preeclampsia; that is, to provide a preeclampsia prognosis.
  • the preeclampsia marker level representation may be used to predict a subject's susceptibility, or risk, of developing preeclampsia.
  • predicting if the individual will develop preeclampsia it is meant determining the likelihood that an individual will develop preeclampsia in the next week, in the next 2 weeks, in the next 3 weeks, in the next 5 weeks, in the next 2 months, in the next 3 months, or during the remainder of the pregnancy.
  • the preeclampsia marker level representation may be used to predict the course of disease progression and/or disease outcome, e.g. expected onset of the preeclampsia, expected duration of the preeclampsia, expectations as to whether the preeclampsia will develop into edampsia, etc.
  • the preeclampsia marker level representation may be used to predict a subject’s responsiveness to treatment for the preeclampsia, e.g., positive response, a negative response, no response at all.
  • Treatment covers any treatment of a disease in a mammal, and indudes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease.
  • the therapeutic agent may be administered before, during or after the onset of disease or injury.
  • the treatment of ongoing disease where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest.
  • the subject therapy may be administered prior to the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
  • the terms "individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • Preedampsia treatments are well known in the art, and may include bed rest, drinking extra water, a low salt diet, medicine to control blood pressure, corticosteroids, inducing pregnancy, and the like.
  • the subject methods of providing a preeclampsia assessment may comprise comparing the obtained preeclampsia marker level representation to a preeclampsia phenotype determination element to identify similarities or differences with the phenotype determination element, where the similarities or differences that are identified are then employed to provide the preeclampsia assessment, e.g.
  • preeclampsia if the subject is healthy or is affeded by preeclampsia, if the subject has a preeclampsia that is likely to progress to eclampsia, if the subject has a preeclampsia that is responsive to therapy, etc.
  • the phenotype determination element may be a positive reference/control, e.g., a sample or marker level representation thereof from a pregnant woman that has preeclampsia, or that will develop preeclampsia, or that has preeclampsia that is manageable by known treatments, or that has preeclampsia that has been determined to be responsive only to the delivery of the baby.
  • the phenotype determination element may be a negative reference/control, e.g. a sample or marker level representation thereof from a pregnant woman that has not developed preeclampsia, or an woman that is not pregnant.
  • Phenotype determination elements are preferably the same type of sample or, if marker level representations, are obtained from the same type of sample as the sample that was employed to generate the marker level representation for the individual being monitored. For example, if the serum of an individual is being evaluated, the phenotype determination element would preferably be of serum.
  • the obtained marker level representation is compared to a single phenotype determination element to obtain information regarding the individual being tested for preeclampsia.
  • the obtained marker level representation is compared to two or more phenotype determination elements.
  • the obtained marker level representation may be compared to a negative reference and a positive reference to obtain confirmed information regarding if the individual will develop preeclampsia.
  • the obtained marker level representation may be compared to a reference that is representative of a preeclampsia that is responsive to treatment and a reference that is representative of a preeclampsia that is not responsive to treatment to obtain information as to whether or not the patient will be responsive to treatment.
  • the comparison of the obtained marker level representation to the one or more phenotype determination elements may be performed using any convenient methodology, where a variety of methodologies are known to those of skill in the art. For example, those of skill in the art of ELISAs will know that ELISA data may be compared by, e.g. normalizing to standard curves, comparing normalized values, etc.
  • the comparison step results in information regarding how similar or dissimilar the obtained marker level profile is to the control/reference profile(s), which simiiarity/dissimiiarity information is employed to, for example, predict the onset of a preeclampsia, diagnose preeclampsia, monitor a preeclampsia patient, etc.
  • array profiles may be compared by, e.g., comparing digital images of the expression profiles, by comparing databases of expression data, etc.
  • Patents describing ways of comparing expression profiles indude, but are not limited to, U.S. Patent Nos. 6,308,170 and 6,228,575, the disclosures of which are herein incorporated by reference. Methods of comparing marker level profiles are also described above. Similarity may be based on relative marker levels, absolute marker levels or a combination of both.
  • a similarity determination is made using a computer having a program stored thereon that is designed to receive input for a marker level representation obtained from a subject, e.g., from a user, determine similarity to one or more reference profiles or reference scores, and return an preeclampsia prognosis, e.g., to a user (e.g., lab technician, physician, pregnant individual, etc.). Further descriptions of computer-implemented aspects of the invention are described below.
  • a similarity determination may be based on a visual comparison of the marker level representation, e.g. preeclampsia score, to a range of phenotype determination elements, e.g.
  • the above comparison step yields a variety of different types of information regarding the cell/bodily fluid that is assayed. As such, the above comparison step can yield a positive/negative prediction of the onset of preeclampsia, a positive/negative diagnosis of preeclampsia, a characterization of a preeclampsia, information on the responsiveness of a preeclampsia to treatment, and the like.
  • the subject methods may be employed for a variety of different types of subjects.
  • the subjects are within the class mammalian, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), lagomorpha (e.g. rabbits) and primates (e.g., humans, chimpanzees, and monkeys).
  • the animals or hosts i.e., subjects (also referred to herein as patients), are humans.
  • the subject methods of providing a preeclampsia assessment include providing a diagnosis, prognosis, or result of the monitoring.
  • the preeclampsia assessment of the present disclosure is provided by providing, i.e. generating, a written report that includes the artisan's assessment, for example, the artisan's determination of whether the patient is currently affected by preeclampsia, of the type, stage, or severity of the subjed's preeclampsia, etc. (a "preeclampsia diagnosis"); the artisan's prediction of the patient's susceptibility to developing preeclampsia, of the course of disease progression, of the patient’s responsiveness to treatment, etc.
  • the subject methods may further include a step of generating or outputting a report providing the results of an artisan's assessment, which report can be provided in the form of an electronic medium (e.g., an electtonic display on a computer monitor), or in the form of a tangible medium (e.g., a report printed on paper or other tangible medium). Any form of report may be provided, e.g. as known in the art or as described in greater detail below.
  • a report providing the results of an artisan's assessment, which report can be provided in the form of an electronic medium (e.g., an electtonic display on a computer monitor), or in the form of a tangible medium (e.g., a report printed on paper or other tangible medium). Any form of report may be provided, e.g. as known in the art or as described in greater detail below.
  • a "report,” as described herein, is an electronic or tangible document which includes report elements that provide information of interest relating to the assessment of a subject and its results.
  • a subject report includes at least a preeclampsia marker representation, e.g. a preeclampsia profile or a preeclampsia score, as discussed in greater detail above.
  • a subject report includes at least an artisan's preeclampsia assessment, e.g. preeclampsia diagnosis, preeclampsia prognosis, an analysis of a preeclampsia monitoring, a treatment recommendation, etc.
  • a subject report can be completely or partially electronically generated.
  • a subject report can further include one or more of: 1 ) information regarding the testing facility; 2) service provider information; 3) patient data; 4) sample data; 5) an assessment report, which can include various information including: a) reference values employed, and b) test data, where test data can include, e.g., a protein level determination; 6) other features.
  • the report may include information about the testing facility, which information is relevant to the hospital, clinic, or laboratory in which sample gathering and/or data generation was conducted.
  • Sample gathering can include obtaining a fluid sample, e.g. blood, saliva, urine etc.; a tissue sample, e.g. a tissue biopsy, etc. from a subject.
  • Data generation can include measuring the marker concentration in preeclampsia patients versus healthy individuals, i.e. individuals that do not have and/or do not develop preeclampsia.
  • This information can include one or more details relating to, for example, the name and location of the testing facility, the identity of the lab technician who conducted the assay and/or who entered the input data, the date and time the assay was conducted and/or analyzed, the location where the sample and/or result data is stored, the lot number of the reagents (e.g., kit, etc.) used in the assay, and the like. Report fields with this information can generally be populated using information provided by the user.
  • the report may include a patient data section, including patient medical history (which can include, e.g., age, race, serotype, prior preeclampsia episodes, and any other characteristics of the pregnancy), as well as administrative patient data such as information to identify the patient (e.g., name, patient date of birth (DOB), gender, mailing and/or residence address, medical record number (MRN), room and/or bed number in a healthcare facility), insurance information, and the like), the name of the patient's physician or other health professional who ordered the monitoring assessment and, if different from the ordering physidan, the name of a staff physician who is responsible for the patient's care (e.g., primary care physician).
  • patient medical history which can include, e.g., age, race, serotype, prior preeclampsia episodes, and any other characteristics of the pregnancy
  • administrative patient data such as information to identify the patient (e.g., name, patient date of birth (DOB), gender, mailing and/or residence address, medical record number (M
  • the report may include an assessment report section, which may include information generated after processing of the data as described herein.
  • the interpretive report can include a prediction of the likelihood that the subject will develop preeclampsia.
  • the interpretive report can include a diagnosis of preeclampsia.
  • the interpretive report can include a characterization of preeclampsia.
  • the assessment portion of the report can optionally also include a recommendation(s). For example, where the results indicate that preeclampsia is likely, the recommendation can include a recommendation that diet be altered, blood pressure medicines administered, etc., as recommended in the art.
  • the reports can include additional elements or modified elements.
  • the report can contain hyperlinks which point to internal or external databases which provide more detailed information about selected elements of the report.
  • the patient data element of the report can include a hyperlink to an electronic patient record, or a site for accessing such a patient record, which patient record is maintained in a confidential database. This latter embodiment may be of interest in an in-hospital system or in-clinic setting.
  • the report is recorded on a suitable physical medium, such as a computer readable medium, e.g., in a computer memory, zip drive, CD, DVD, etc.
  • reagents, systems and kits thereof for practicing one or more of the above-described methods.
  • the subject reagents, systems and kits thereof may vary greatly.
  • Reagents of interest include reagents specifically designed for use in producing the above- described marker level representations of preeclampsia markers from a sample, for example, one or more detection elements, e.g. antibodies or peptides for the detection of protein, oligonucleotides for the detection of nucleic acids, etc.
  • the detection element comprises a reagent to detect the expression of a single preeclampsia marker
  • the detection element may be a dipstick, a plate, an array, or cocktail that comprises one or more detection elements, e.g. one or more antibodies, one or more oligonucleotides, one or more sets of PCR primers, etc. which may be used to detect the expression of one or more preeclampsia marker simultaneously,
  • Another type of such reagent is an array of probe nucleic acids in which the genes of interest are represented.
  • array formats are known in the art, with a wide variety of different probe structures, substrate compositions and attachment technologies (e.g., dot blot arrays, microarrays, etc.).
  • Representative array structures of interest include those described in U.S. Patent Nos.: 5,143,854; 5,288,644; 5,324,633;
  • probes, collections of primers, or collections of antibodies that include probes, primers or antibodies (also called reagents) that are specific for at least 1 gene/protein/lipd selected from the group consisting of LEP, Ceramide (d 18: 1/25:0), Ceramide (d18: 1/26:0), or a biochemical substrate specific for the cofactor/prosthetic group heme.
  • the collection of probes, primers, or antibodies includes reagents specific for LEP, Ceramide (d18:1/25:0), Ceramide (d 18: 1/26:0) as well as a biochemical substrate specific for heme.
  • the subject probe, primer, or antibody collections or reagents may include reagents that are specific only for the genes/proteins/lipids/cofactors that are listed above, or they may include reagents specific for additional genes/proteins/lipids/cofactors that are not listed above, such as probes, primers, or antibodies specific for genes/proteins/lipids/cofactors whose expression pattern are known in the art to be associated with preeclampsia, e.g. and sFlt-1 (VEGF-RI) and PIGF.
  • preeclampsia e.g. and sFlt-1 (VEGF-RI) and PIGF.
  • a system may be provided.
  • system refers to a collection of reagents, however compiled, e.g., by purchasing the collection of reagents from the same or different sources.
  • kit refers to a collection of reagents provided, e.g., sold, together.
  • the nucleic acid- or antibody-based detection of the sample nucleic add or protein, respectively may be coupled with an electrochemical biosensor platform that will allow multiplex determination of these biomarkers for personalized preeclampsia care.
  • hybridization and washing buffers prefabricated probe arrays, labeled probe purification reagents and components, like spin columns, etc.
  • signal generation and detection reagents e.g. labeled secondary antibodies, streptavidin-alkaline phosphatase conjugate, chemifluorescent or chemiluminescent substrate, and the like.
  • the subject systems and kits may also indude one or more preeclampsia phenotype determination elements, which element is, in many embodiments, a reference or control sample or marker representation that can be employed, e.g., by a suitable experimental or computing means, to make a preeclampsia prognosis based on an "input" marker level profile, e.g., that has been determined with the above described marker determination element.
  • Representative preeclampsia phenotype determination elements include samples from an individual known to have or not have preeclampsia, databases of marker level representations, e.g., reference or control profiles or scores, and the like, as described above.
  • the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc.
  • Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded.
  • Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.
  • PE is a multisystem disorder of pregnancy with the placenta playing a pivotal role.
  • Investigators have used genetic, genomic, proteomic, and lipidomic approaches to compare PE and control placental tissues.
  • Transcriptional profiling of case-control samples has identified disease-specific expression patterns, canonical pathways and gene-gene networks (Lapaire et al. Microarray screening for novel preeclampsia biomarker candidates. Fetal diagnosis and therapy 2012;31:147-53; Nishizawa et al. Microarray analysis of differentially expressed fetal genes in placenta tissue derived from early and late onset severe preeclampsia.
  • Transcriptional profiling of human placentas from pregnancies complicated by preeclampsia reveals disregulation of sialic acid acetylesterase and immune signaling pathways.
  • Placenta 2011;32:175-82; Winn et al. Severe preeclampsia-related changes in gene expression at fee maternal-fetal interlace include sialic acid-binding immunoglobulin-like lectin-6 and pappalysin-2. Endocrinology 2009;150:452-62).
  • Preeclampsia-related biomarker studies Kolia et al. Quantitative proteomic (iTRAQ) analysis of 1st trimester maternal plasma samples in pregnancies at risk for preeclampsia.
  • Placental angiogenic and anti-angiogenic factor imbalance elevated soluble fms-like tyrosine kinase (sFlt-1 ) and decreased placental growth factor (PIGF) levels, are suggested in the pathogenesis of PE (Shibata et al. Soluble fms-like tyrosine kinase 1 is increased in preeclampsia but not in normotensive pregnancies with small-for-gestational-age neonates: relationship to circulating placental growth factor. The Journal of clinical endocrinology and metabolism 2005;90:4895-903; Maynard et al.
  • sFlt-1 Excess placental soluble fms-like tyrosine kinase 1 (sFlt-1 ) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.
  • vascular endothelial growth factor receptor- 1 (Flt-1 ) and soluble Flt-1 (sFlt-1), by peripheral blood mononuclear cells (PBMCs) in normotensive and preeclamptic pregnant women.
  • PBMCs peripheral blood mononuclear cells
  • PLoS computational biology 2010;6 in meta-analysis allowed us to identify consistent and significant differential gene expression across experiments to develop biomarkers for downstream experimental validation.
  • Serum proteins are routinely used to diagnose diseases, but sensitive and specific biomarkers are hard to find and may be due to their low serological abundance, which can easily be masked by highly abundant proteins.
  • Our serum protein marker discovery method (Ling et al. Plasma profiles in active systemic juvenile idiopathic arthritis: Biomarkers and biological implications. Proteomics 2010) combines antibody-based serum abundant protein depletion and 2D gel comparative profiling to discover differential protein gel spots between PE and control sera for subsequent protein mass spectrometric identification. We hypothesized that there would be differential serological signatures allowing PE diagnosis.
  • ELISA was performed on samples using commercial kits following vendors' instructions. Assays were performed to measure serum level of LEP, sFlt-1, and PIFG. Two types of ELISA were applied, sandwich ELISA and competitive ELISA. Briefly, for sandwich ELISA, the capture antibody has been pre-coated onto the microplate and then standards and serum samples are added into the wells of the microplate to bind with the capture antibody. After extensive washing to avoid nonspecific binders, a second detection antibody conjugated with horseradish peroxidase (HRP) was added to the wells.
  • HRP horseradish peroxidase
  • a HRP substrate solution followed by stop solution is added to the microplate wells.
  • the optical density (O.D.) of the microplate wells is measured and the O.D. is proportional to the amount of analyte present in the sample.
  • the sample analyte concentration is calculated based on standard curve.
  • the capture antibody for antiserum is pre-coated onto the microplate.
  • a constant concentration of biotinylated tracer (Bt-tracer) and varying concentrations of unlabeled standard or sample peptide are added into the wells and they compete for binding specifically to the antiserum.
  • streptavidin-conjugated HRP is added into the wells to bind Bt-tracer specifically, which produces a soluble colored product after a substrate is added.
  • the optical density (O.D.) of the microplate wells is measured and the O.D. is inverse proportional to the amount of analyte present in the sample.
  • Step 1 Materials.
  • the calibration standard ceramide (d18: 1/24:0) and stable isotope labeled internal standards d7-ceramide (d18:1/24:0) were purchased from Avanti Lipids (Alabaster, AL).
  • HPLC grade water, methanol, 2-propanol, and chloroform were obtained from Fisher Scientific (Pittsburgh, PA).
  • Analytical grade ammonium bicarbonate was purchased from Sigma Aldrich (St. Louis, MO).
  • the de-lipidized serum VD-DDC Mass Spec Gold was obtained from Golden West Biological (Temecula, CA). All materials were directly used without further purification.
  • Step 2 MRM Transition Optimization.
  • the MRM transitions for targeted ceramides and dihydroceramides were individually optimized by direct syringe pump infusion of 0.50 uM of the corresponding standard at 10 ⁇ L/min into the mass spectrometer in the presence of 10 mM of ammonium bicarbonate.
  • the SRM transitions were optimized and recorded for parent ion m/z, daughter ion m/z, collision energy, and RF lens on a Thermo TSQ Quantiva mass spectrometer.
  • the Q1 and Q3 resolutions were both set at 0.7 Da.
  • Step 3 Sample Preparation.
  • 10- ⁇ L aliquot of de-lipidized serum was spiked with 10 ⁇ L of 2-propanol to obtain blank sample.
  • the blank samples were extracted with 200 ⁇ L of methanol and internal standard working solution to obtain double and single blanks, respectively.
  • 10- ⁇ L aliquot of de-lipidized serum was spiked with 10 ⁇ L of calibrator working solution to obtain the calibrator at the corresponding level.
  • the spiked calibrators were individually extracted with 200 ⁇ L of internal standard working solution to obtain a set of calibrators based on 6 concentration levels.
  • the retention time-dependent data acquisition was employed using pre-defined retention time windows with variable widths (1.2 mins for medium drain and 1.5 mins for long chain ceramides and dihydroceramides) to record the extracted ion chromatograms (EIC) of targeted analytes.

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Abstract

L'invention concerne des marqueurs de la préclampsie, des panels de marqueurs de la préclampsie, et des méthodes d'obtention d'une représentation du niveau de marqueurs de la préclampsie dans un échantillon. Ces compositions et méthodes sont utilisables dans un certain nombre d'applications incluant, par exemple, le diagnostic de la préclampsie, le pronostic de la préclampsie, la surveillance d'un sujet atteint de préclampsie, et la détermination d'un traitement pour la préclampsie. L'invention concerne en outre des systèmes, des dispositifs et des kits associés, utilisables dans la mise en pratique desdites méthodes.
PCT/US2021/020417 2021-03-02 2021-03-02 Méthodes et compositions pour fournir une évaluation de prééclampsie à l'aide de leptine et de céramide WO2022186821A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150099655A1 (en) * 2012-05-08 2015-04-09 The Board Of Trustees Of The Leland Stanford Junior University Methods and Compositions for Providing a Preeclampsia Assessment
WO2019197838A1 (fr) * 2018-04-12 2019-10-17 Oxford University Innovation Limited Biomarqueurs et utilisations de ces derniers

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150099655A1 (en) * 2012-05-08 2015-04-09 The Board Of Trustees Of The Leland Stanford Junior University Methods and Compositions for Providing a Preeclampsia Assessment
WO2019197838A1 (fr) * 2018-04-12 2019-10-17 Oxford University Innovation Limited Biomarqueurs et utilisations de ces derniers

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HUANG QIANYANG, HAO SHIYING, YAO XIAOMING, YOU JIN, LI XIAO, LAI DONGHAI, HAN CHUNLE, SCHILLING JAMES, HWA KUO YUAN, THYPARAMBIL S: "Quantitative LCMS for ceramides/dihydroceramides: pregnancy baseline biomarkers and potential metabolic messengers", BIORXIV, 25 February 2020 (2020-02-25), pages 1 - 52, XP055967866, [retrieved on 20221004], DOI: 10.1101/2020.02.24.963462 *
HUANG QIANYANG, SHIYING HAO, JIN YOU, XIAOMING YAO, ZHEN LI, JAMES SCHILLING, ZHEN LI, SHEENO THYPARAMBIL: "Case finding of early pregnancies at risk of preeclampsia using maternal blood leptin/ceramide ratio: multi-omics discovery and validation from a longitudinal study", MEDRXIV, 7 January 2021 (2021-01-07), pages 1 - 33, XP055967859, Retrieved from the Internet <URL:http://dx.doi.org/10.1101/ 2020.12.17.20248418> [retrieved on 20221004], DOI: 10.1101/ 2020.12.17.20248418 *

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