WO2018076134A1 - Procédés et trousses destinés à l'estimation de pré-éclampsie et au pronostic de naissance prématurée - Google Patents

Procédés et trousses destinés à l'estimation de pré-éclampsie et au pronostic de naissance prématurée Download PDF

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WO2018076134A1
WO2018076134A1 PCT/CN2016/103060 CN2016103060W WO2018076134A1 WO 2018076134 A1 WO2018076134 A1 WO 2018076134A1 CN 2016103060 W CN2016103060 W CN 2016103060W WO 2018076134 A1 WO2018076134 A1 WO 2018076134A1
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preeclampsia
markers
preterm birth
panel
subject
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PCT/CN2016/103060
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English (en)
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Wenkai XIANG
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Ldx Prognostics Limited Co.
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Priority to CN201680090000.2A priority Critical patent/CN109891239B/zh
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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
    • G01N33/68—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
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/20—Supervised data analysis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients

Definitions

  • This disclosure pertains to methods and kits for providing a preeclampsia assessment and prognosing preterm birth.
  • Preeclampsia is a serious multisystem complication of pregnancy with adverse effects for mothers and babies.
  • the incidence of the disorder is around 5-8%of all pregnancies in the U.S. and worldwide, and the disorder is responsible for 18%of all maternal deaths in the U.S.
  • the causes and pathogenesis of preeclampsia remain uncertain, and the diagnosis relies on nonspecific laboratory and clinical signs and symptoms that occur late in the disease process, sometimes making the diagnosis and clinical management decisions difficult.
  • Earlier and more reliable disease diagnosing, prognosing and monitoring will lead to more timely and personalized preeclampsia treatments and significantly advance our understanding of preeclampsia pathogenesis.
  • Preeclampsia/preterm birth markers preeclampsia/preterm birth marker panels, and methods for differentiating among a normal subject, a preeclampsia subject and a preterm birth subject are provided. These methods find use in a number of applications, including, for example, diagnosing preeclampsia, prognosing a preeclampsia or preterm birth, monitoring a subject with preeclampsia or with risk of preterm birth, and determining a treatment for preeclampsia or preterm birth.
  • systems, devices and kits thereof that find use in practicing the subject methods are provided.
  • a method for providing a preeclampsia assessment and prognosing preterm birth for a specific subject comprises (i) developing and training a random forest model using a plurality of clinical and laboratory test variables of randomly selected subjects, in order to derive a scoring metric to differentiate among a normal subject, a preeclampsia subject and a preterm birth subject; (ii) evaluating a panel of markers from the specific subject to determine the level of each of the panel of markers; and (iii) feeding the level of each of the panel of markers into the random forest model to provide the preeclampsia assessment and prognose preterm birth, wherein the panel of markers comprises inhibin beta A (Activin A) and at least one selected from a group consisting of ADAM metallopeptidase domain 12 (Adam12) , body mass index (BMI) and white blood cell count (WBC) .
  • Activin A inhibin beta A
  • WBC white blood cell count
  • the plurality of clinical and laboratory test variables comprises at least two variables selected from a group consisting of systolic blood pressure, diastolic blood pressure, Activin A, gestational age, proteinuria, preeclampsia history, white blood cell count, preterm times, number of full-term pregnancy, dducation, ADAM12, multiple pregnancy, maternal height, maternal weight, BMI, abortion time, and age at blood collection.
  • the plurality of clinical and laboratory test variables comprises inhibin beta A (Activin A) and at least one selected from a group consisting of ADAM metallopeptidase domain 12 (Adam12) , body mass index (BMI) and white blood cell count (WBC) .
  • a panel of preeclampsia and preterm birth markers comprising inhibin beta A (Activin A) and one or more markers selected from the group consisting of Adam12 (ADAM metallopeptidase domain 12) , body mass index (BMI) and white blood cell count (WBC) .
  • Activin A inhibin beta A
  • BMI body mass index
  • WBC white blood cell count
  • the methods do not include measurement of expression levels of FSTL3, APLN, LEP, INHA, PIK3CB, SLC2A1, CRH, HSD17B1, SIGLEC6, PVRL4, HEXB, IL1 RAP, MFAP5, HTRA1, EBI3, HTRA4.
  • the methods do not include measurement of expression levels of FN1, PEG10, EPAS1, F5, FBN1, HGF, IGF2, AGO2, ATF2, KDM6A, KRAS, MECOM, PDPK1, S100A8, SPTBN1, TRA2B, VEGFA, WNK1, ACSS1, BMP7, CGB, CYP19A1, DLX4, ELOVL2, EZR, HBB, IL6ST, MFSD2A, PEG3, and/or SVEP1.
  • the methods of the present disclosure do not include measurement of expression levels of PAPPA2.
  • the methods do not include measurement of the expression level of FSTL3. In one embodiment, the methods do not include measurement of the expression level of APLN. In one embodiment, the methods do not include measurement of the expression level of LEP. In one embodiment, the methods do not include measurement of the expression level of INHA. In one embodiment, the methods do not include measurement of the expression level of PIK3CB. In one embodiment, the methods do not include measurement of the expression level of SLC2A1. In one embodiment, the methods do not include measurement of the expression level of CRH. In one embodiment, the methods do not include measurement of the expression level of HSD17B1. In one embodiment, the methods do not include measurement of the expression level of SIGLEC6.
  • the methods do not include measurement of the expression level of PVRL4. In one embodiment, the methods do not include measurement of the expression level of HEXB. In one embodiment, the methods do not include measurement of the expression level of IL1 RAP. In one embodiment, the methods do not include measurement of the expression level of MFAP5. In one embodiment, the methods do not include measurement of the expression level of HTRA1. In one embodiment, the methods do not include measurement of the expression level of EBI3. In one embodiment, the methods do not include measurement of the expression level of HTRA4. In one embodiment, the methods do not include measurement of the expression level of F5. In one embodiment, the methods do not include measurement of the expression level of FBN1. In one embodiment, the methods do not include measurement of the expression level of HGF.
  • the methods do not include measurement of the expression level of IGF2. In one embodiment, the methods do not include measurement of the expression level of AGO2. In one embodiment, the methods do not include measurement of the expression level of ATF2. In one embodiment, the methods do not include measurement of the expression level of KDM6A. In one embodiment, the methods do not include measurement of the expression level of KRAS. In one embodiment, the methods do not include measurement of the expression level of MECOM. In one embodiment, the methods do not include measurement of the expression level of PDPK1. In one embodiment, the methods do not include measurement of the expression level of S100A8. In one embodiment, the methods do not include measurement of the expression level of SPTBN1. In one embodiment, the methods do not include measurement of the expression level of TRA2B.
  • the methods do not include measurement of the expression level of VEGFA. In one embodiment, the methods do not include measurement of the expression level of WNK1. In one embodiment, the methods do not include measurement of the expression level of ACSS1. In one embodiment, the methods do not include measurement of the expression level of BMP7. In one embodiment, the methods do not include measurement of the expression level of CGB. In one embodiment, the methods do not include measurement of the expression level of CYP19A1. In one embodiment, the methods do not include measurement of the expression level of DLX4. In one embodiment, the methods do not include measurement of the expression level of ELOVL2. In one embodiment, the methods do not include measurement of the expression level of EZR. In one embodiment, the methods do not include measurement of the expression level of HBB.
  • the methods do not include measurement of the expression level of IL6ST. In one embodiment, the methods do not include measurement of the expression level of MFSD2A. In one embodiment, the methods do not include measurement of the expression level of PEG3. In one embodiment, the methods do not include measurement of the expression level of SVEP1.
  • 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 methods of the present disclosure are suitable for women at different stages of pregnancy, which is unexpected given typically such prognosis is only made for women that are pregnant for more than 32 weeks.
  • the woman is pregnant for 16-27 weeks.
  • the woman is pregnant for 28-31 weeks.
  • the woman is pregnant for 16-31 weeks.
  • the woman is pregnant for less than 32 weeks.
  • the woman is pregnant for 32-36 weeks.
  • the methods may be particularly suitable for certain pregnant women, such as those that smoke or consume alcohol, are younger than 17 or older than 35, have preterm birth history and/or are stressed or unhealthy.
  • 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.
  • the woman can be subject to a procedure that helps ameliorate the preterm birth risk.
  • procedures include, without limitation, administration of corticosteroid, magnesium sulfate, an antibiotic, or progestin, and cervical cerclage and combinations thereof.
  • a kit for making a preeclampsia and preterm birth assessment for a sample.
  • the preeclampsia assessment is a preeclampsia diagnosis.
  • the preterm birth assessment is a preterm birth prognosis.
  • the kit comprises one or more detection elements for measuring the amount of marker in a sample for a panel of preeclampsia and preterm birth markers comprising inhibin beta A (Activin A) .
  • the kit comprises one or more detection elements for measuring the amount of marker in a sample for a panel of preeclampsia and preterm birth markers comprising inhibin beta A (Activin A) and Adam12 (ADAM metallopeptidase domain 12) .
  • the kit comprises one or more detection elements for measuring the amount of marker in a sample for a panel of preeclampsia and preterm birth markers comprising inhibin beta A (Activin A) and body mass index (BMI) .
  • the kit comprises one or more detection elements for measuring the amount of marker in a sample for a panel of preeclampsia and preterm birth markers comprising inhibin beta A (Activin A) and white blood cell count (WBC) .
  • the kit further comprises a preeclampsia phenotype determination element.
  • the kit comprises one or more detection elements for measuring the amount of marker in a sample for a panel of preeclampsia and preterm birth markers comprising one or more markers selected from the group consisting of inhibin beta A (Activin A) and Adam12 (ADAM metallopeptidase domain 12) ; and a preeclampsia and preterm birth phenotype determination element.
  • the one or more detection elements detect the level of marker polypeptides in the sample.
  • Figure 1 Study outline of the multi- ‘omics’ , based discovery and validation of PE biomarkers.
  • Figure 3 Identification of PE biomarkers using a combination of meta-analysis, protein atlas analysis, and human orthologues analysis.
  • Figure 4 Identification of PTB biomarkers using a combination of meta-analysis, protein atlas analysis, and human orthologues analysis.
  • FIG. 1 Expression comparative analysis of PE biomarkers (PE versus controls) .
  • Forest plot summarizes the results of placenta mRNA expression meta analysis, and maternal serum analyte abundance quantification at different early and late gestational age weeks. Line plot represents 95%confidence interval.
  • Figure 6 Transcription analysis of the candidate genes for preterm birth.
  • Placenta gene expression unit: FPKM
  • middle panel gene expression ratio between placenta and other organ tissues
  • right panel gene expression ratio of the placenta tissue between preterm birth and normal controls.
  • Figure 7 Boxplot display and scatter plot of biomarker distribution for Activin A at different gestational age weeks at blood sample collection in PE, PTB, and control groups. Horizontal box boundaries and midline denote sample quartiles. B: Scatter plots of biomarker distribution for Activin A as a function of gestational age weeks at blood sample collection (Top) , delivery (Bottom) , and the gap in between (Middle) .
  • Figure 8. Boxplot display and scatter plot of biomarker distribution for Adam12 at different gestational age weeks at blood sample collection in PE, PTB, and control groups. Horizontal box boundaries and midline denote sample quartiles.
  • B Scatter plots of biomarker distribution for Adam12 as a function of gestational age weeks at blood sample collection (Top) , delivery (Bottom) , and the gap in between (Middle) .
  • FIG. 9 A: Score distribution for each sample. B: Importance of each feature in the algorithm. The scores and feature importance were produced by a random forest algorithm developed with a panel of Activin A and Adam12.
  • FIG. 10 A: Score distribution for each sample. B: Importance of each feature in the algorithm. The scores and feature importance were produced by a random forest algorithm developed with a panel of Activin A and BMI.
  • FIG. 11 A: Score distribution for each sample. B: Importance of each feature in the algorithm. The scores and feature importance were produced by a random forest algorithm developed with a panel of Activin A and white blood counts.
  • Preeclampsia and preterm birth markers, preeclampsia and preterm birth marker panels, and methods for obtaining a preeclampsia and preterm birth 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 or preterm birth, monitoring a subject with preeclampsia or risks of preterm birth, and determining a treatment for preeclampsia and preterm birth.
  • 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 and preterm birth assessment, e.g. diagnosing, prognosing, monitoring, and/or treating preeclampsia and/or preterm birth 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-normal liver enzymes, and thrombocytopenia.
  • Preeclampsia typically occurs in the third trimester of pregnancy, but in severe cases, the disorder occur 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.
  • Preterm birth or “spontaneous preterm birth” refers to preterm birth, also known as premature birth, which is the birth of a baby at less than 37 weeks gestational age. These babies are known as preemies or premmies. Symptoms of preterm labor include uterine contractions which occur more often than every ten minutes or the leaking of fluid from the vagina. Premature infants are at greater risk for cerebral palsy, delays in development, hearing problems, and problems seeing. These risks are greater the earlier a baby is born.
  • diagnosing a preeclampsia/preterm birth or “providing a preeclampsia/preterm birth diagnosis, “it is generally meant providing a preeclampsia/preterm birth determination, e.g. a determination as to whether a subject (e.g.
  • a subject that has clinical symptoms of preeclampsia/preterm birth, a subject that is asymptomatic for preeclampsia/preterm birth but has risk factors associated with preeclampsia, a subject that is asymptomatic for preeclampsia and has no risk factors associated with preeclampsia/preterm birth) is presently affected by preeclampsia; a classification of the subject's preeclampsia/preterm birth into a subtype of the disease or disorder; a determination of the severity of preeclampsia/preterm birth; and the like.
  • preeclampsia/preterm birth or “providing a preeclampsia/preterm birth prognosis, “it is generally meant providing a preeclampsia/preterm birth prediction, e.g. a prediction of a subject's susceptibility, or risk, of developing preeclampsia/preterm birth; a prediction of the course of disease progression and/or disease outcome, e.g. expected onset of the preeclampsia/preterm birth, expected duration of the preeclampsia, expectations as to whether the preeclampsia will develop into eclampsia, etc.
  • a preeclampsia/preterm birth prediction e.g. a prediction of a subject's susceptibility, or risk, of developing preeclampsia/preterm birth
  • a prediction of the course of disease progression and/or disease outcome e.g. expected onset of the preeclampsia/preterm birth, expected duration
  • a prediction of a subject's responsiveness to treatment for the preeclampsia/preterm birth e.g., positive response, a negative response, no response at all; and the like.
  • monitoring a preeclampsia/preterm birth, it is generally meant monitoring a subject's condition, e.g. to inform a preeclampsia/preterm birth diagnosis, to inform a preeclampsia/preterm birth prognosis, to provide information as to the effect or efficacy of a preeclampsia/preterm birth treatment, and the like.
  • treating a preeclampsia/preterm birth it is meant prescribing or providing any treatment of a preeclampsia/preterm birth in a mammal, and includes: (a) preventing the preeclampsia/preterm birth from occurring in a subject which may be predisposed to preeclampsia/preterm birth but has not yet been diagnosed as having it; (b) inhibiting the preeclampsia/preterm birth, i.e., arresting its development; or (c) relieving the preeclampsia/preterm birth, i.e., causing regression of the preeclampsia/preterm birth.
  • “Prognosis” as used herein generally includes a prediction of a subject’s susceptibility to a disease or disorder, i.e. preterm birth; a determination, or diagnosis, as to whether a subject is presently affected by a disease or disorder, i.e. preterm birth; a prediction for a subject affected by a disease or disorder (e.g., determination of the severity of preterm birth, likelihood that a preterm birth condition will develop into early delivery) ; a prediction of a subject’s responsiveness to treatment for the disease or disorder; and the monitoring a subject’s condition to provide information as to the effect or efficacy of therapy.
  • treatment covers any treatment of a disease in a mammal, and includes: (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. Such treatment is desirably performed prior to complete loss of function in the affected tissues.
  • the subject therapy will desirably be administered during 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.
  • the body mass index (BMI) , also known as Quetelet index, is a value derived from the mass (weight) and height of an individual.
  • the BMI is defined as the body mass divided by the square of the body height, and is universally expressed in units of kg/m 2 , resulting from mass in kilograms and height in metres.
  • the BMI is an attempt to quantify the amount of tissue mass (muscle, fat, and bone) in an individual, and then categorize that person as underweight, normal weight, overweight, or obese based on that value. Commonly accepted BMI ranges are underweight: under 18.5 kg/m 2 , normal weight: 18.5 to 25, overweight: 25 to 30, obese: over 30.
  • WBCs White blood cells
  • CBC complete blood count
  • compositions useful for providing a preeclampsia assessment will be described first, followed by methods, systems and kits for their use.
  • preeclampsia and preterm birth markers and panels of preeclampsia and preterm birth markers are provided.
  • a preeclampsia and preterm birth marker it is meant a molecular entity whose representation in a sample is associated with a preeclampsia phenotype or preterm birth phenotype.
  • a preeclampsia and preterm birth marker may be differentially represented, i.e.
  • an elevated level of marker is associated with the preeclampsia phenotype and/or preterm birth phenotype.
  • the concentration of marker in a sample may be 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 7.5-fold, 10-fold, or greater in a sample associated with the preeclampsia phenotype or preterm birth phenotype than in a sample not associated with the preeclampsia phenotype or preterm phenotype; or the concentration of marker in a sample may be 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 7.5-fold, 10-fold, or greater in a sample associated with the preeclampsia phenotype than in a sample associated with preterm phenotype.
  • a reduced level of marker is associated with the preeclampsia phenotype or preterm birth phenotype.
  • the concentration of marker in a sample may be 10%less, 20%less, 30%less, 40%less, 50%less or more in a sample associated with the preeclampsia or preterm birth phenotype than in a sample not associated with the preeclampsia or preterm birth phenotype; or the concentration of marker in a sample may be 10%less, 20%less, 30%less, 40%less, 50%less or more in a sample associated with the preeclampsia phenotype than in a sample associated with the preterm birth phenotype.
  • Preeclampsia and preterm birth markers may include proteins associated with preeclampsia, preterm birth, and their corresponding genetic sequences, i.e. mRNA, DNA, etc.
  • a “gene” or “recombinant gene” it is meant a nucleic acid comprising an open reading frame that encodes for the protein.
  • a coding sequence The boundaries of a coding sequence are determined by a start codon at the 5'(amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a transcription termination sequence may be located 3'to the coding sequence.
  • a gene may optionally include its natural promoter (i.e., the promoter with which the exons and introns of the gene are operably linked in a non-recombinant cell, i.e., a naturally occurring cell) , and associated regulatory sequences, and may or may not have sequences upstream of the AUG start site, and may or may not include untranslated leader sequences, signal sequences, downstream untranslated sequences, transcriptional start and stop sequences, polyadenylation signals, translational start and stop sequences, ribosome binding sites, and the like.
  • the inventors have identified a number of molecular entities that are associated with preeclampsia or preterm birth and that find use in combination (i.e. as a panel) in providing a preeclampsia and preterm birth assessment, e.g. diagnosing preeclampsia, prognosing a preeclampsia and preterm birth, monitoring a subject with preeclampsia and/or preterm birth, determining a treatment for a subject affected with preeclampsia or preterm birth, and the like.
  • these include, but are not limited to, inhibin beta A (Activin A, Genbank Accession No.
  • ADAM metallopeptidase domain 12 (Adam12, GenBank Accession No. NP_001275903.1, NM_001288974.1 [O43184-4] ; NP_001275904.1. NM_001288975.1. [O43184-3] , NP_003465.3, NM_003474.5, [O43184-1] , NP_067673.2. NM_021641.4. [O43184-2] ) .
  • preeclampsia and preterm birth panels are preeclampsia and preterm birth panels.
  • a “panel” of preeclampsia and preterm birth markers it is meant two or more preeclampsia and preterm birth markers, e.g. 3 or more, 4 or more, or 5 or more markers, whose levels, when considered in combination, find use in providing a preeclampsia and preterm birth assessment, e.g. making a preeclampsia and preterm birth risk diagnosis, prognosis, monitoring, and/or treatment.
  • the preeclampsia panel may comprise Activin A and Adam12.
  • preeclampsia and preterm birth markers that find use as preeclampsia panels in the subject methods may be readily identified by the ordinarily skilled artisan using any convenient statistical methodology, e.g. as known in the art or described in the working examples herein.
  • the panel of analytes may be selected by combining genetic algorithm (GA) and all paired (AP) support vector machine (SVM) methods for preeclampsia classification analysis.
  • G genetic algorithm
  • AP all paired
  • SVM support vector machine
  • Predictive features are automatically determined, e.g. through iterative GA/SVM, leading to very compact sets of non-redundant preeclampsia-relevant analytes with the optimal classification performance. While different classifier sets will typically harbor only modest overlapping gene features, they will have similar levels of accuracy in providing a preeclampsia assessment to those described above and in the working examples herein.
  • methods are provided for providing a preeclampsia assessment and prognosing preterm birth for a specific subject.
  • a random forest model needs to be developed and trained using a plurality of clinical and laboratory test variables of randomly selected subjects, in order to derive a scoring metric to differentiate among a normal subject, a preeclampsia subject and a preterm birth subject.
  • a random forest model is a model generated through the random forest algorithm.
  • Random forests or random decision forests are an ensemble learning method for classification, regression and other tasks, that operate by constructing a multitude of decision trees at training time and outputting the class that is the mode of the classes (classification) or mean prediction (regression) of the individual trees.
  • the first algorithm for random decision forests was created by Tin Kam Ho using the random subspace method which, in Ho's formulation, is a way to implement the "stochastic discrimination" approach to classification proposed by Eugene Kleinberg.
  • An extension of the algorithm was developed by Leo Breiman and Adele Cutler and "Random Forests" is their trademark. The extension combines Breiman's "bagging" idea and random selection of features, introduced first by Ho and later independently by Amit and Geman in order to construct a collection of decision trees with controlled variance.
  • a “forest” comprises a plurality of binary “trees” , and at each node, “trees” were split by choosing a split variable value producing the maximum node separation. “Trees” were constructed until each of the terminal nodes reached a sample size of 1. Final decisions were reached by averaging the decisions of each tree (Breiman L. Random forests. Machine Learning 2001; 45: 5-32; Breiman L. Bagging predictors. Machine Learning 1996; 24: 123-40. ) .
  • An exemplary random forest model was constructed in a literature (See, e.g., Shiying Hao et. al. Classification Tool for Differentiation of Kawasaki Disease from Other Febrile Illnesses, The Journal of Pediatrics, Volume 176, September 2016, Pages 114–120. e8) .
  • the plurality of clinical and laboratory test variables comprises at least two variables selected from physiological and/or biochemical factors, including but are not limited to systolic blood pressure, diastolic blood pressure, Activin A, gestational age, proteinuria, preeclampsia history, white blood cell count, preterm times, number of full-term pregnancy, education, ADAM12, multiple pregnancy, maternal height, maternal weight, BMI, abortion time, and age at blood collection.
  • physiological and/or biochemical factors including but are not limited to systolic blood pressure, diastolic blood pressure, Activin A, gestational age, proteinuria, preeclampsia history, white blood cell count, preterm times, number of full-term pregnancy, education, ADAM12, multiple pregnancy, maternal height, maternal weight, BMI, abortion time, and age at blood collection.
  • the plurality of clinical and laboratory test variables comprises inhibin beta A (Activin A) and at least one selected from a group consisting of ADAM metallopeptidase domain 12 (Adam12) , body mass index (BMI) and white blood cell count (WBC) .
  • Activin A inhibin beta A
  • Adam12 ADAM metallopeptidase domain 12
  • BMI body mass index
  • WBC white blood cell count
  • biological sample encompasses a variety of sample types obtained from an organism and can be used in a diagnostic, prognostic, or monitoring assay.
  • the term encompasses blood and other liquid samples of biological origin or cells derived therefrom and the progeny thereof.
  • the term encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components.
  • the term encompasses a clinical sample, and also includes cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.
  • Clinical samples for use in the methods of the invention may be obtained from a variety of sources, particularly blood samples.
  • the sample is a serum or serum-derived sample. Any convenient methodology for producing a fluid serum sample may be employed.
  • the method employs drawing venous blood by skin puncture (e.g., finger stick, venipuncture) into a clotting or serum separator tube, allowing the blood to clot, and centrifuging the serum away from the clotted blood. The serum is then collected and stored until assayed. Once the patient derived sample is obtained, the sample is assayed to determine the level of preeclampsia marker (s) .
  • the subject sample is typically obtained from the individual during the second or third trimester of gestation.
  • digstation it is meant the duration of pregnancy in a mammal, i.e. the time interval of development from fertilization until birth, plus two weeks, i.e. to the first day of the last menstrual period.
  • second or third trimester it is meant the second or third portions of gestation, each segment being 3 months long.
  • first trimester is meant from the first day of the last menstrual period through the 13th week of gestation
  • second trimester it is meant from the 14th through 27th week of gestation
  • third trimester it is meant from the 28th week through birth, i.e.
  • a subject sample may be obtained at about weeks 14 through 42 of gestation, at about weeks 18 through 42 of gestation, at about weeks 20 through 42 of gestation, at about weeks 24 through 42 of gestation, at about weeks 30 through 42 of gestation, at about weeks 34 through 42 of gestation, at about weeks 38 through 42 of gestation.
  • the subject sample may be obtained early in gestation, e.g. at week 14 or more of gestation, e.g. at week 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 or more of gestation, more often at week 24, 25, 26, 27, 28, 29, 30, 31, 32, or week 33 or more of gestation.
  • the subject sample may be obtained late in gestation, for example, at or after 34 weeks of gestation, e.g. at week 35, 36, 37, 38, 39, 40, or week 41 of gestation.
  • a sample Once a sample is obtained, it can be used directly, frozen, or maintained in appropriate culture medium for short periods of time.
  • the samples will be from human patients, although animal models may find use, e.g. equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc. Any convenient tissue sample that demonstrates the differential representation in a patient with preeclampsia of the one or more preeclampsia markers disclosed herein may be evaluated in the subject methods.
  • a suitable sample source will be derived from fluids into which the molecular entity of interest, i.e. the RNA transcript or protein, has been released.
  • the subject sample may be treated in a variety of ways so as to enhance detection of the one or more preeclampsia markers.
  • the red blood cells may be removed from the sample (e.g., by centrifugation) prior to assaying.
  • Such a treatment may serve to reduce the non-specific background levels of detecting the level of a preeclampsia and preterm birth marker using an affinity reagent.
  • Detection of a preeclampsia and preterm birth marker may also be enhanced by concentrating the sample using procedures well known in the art (e.g.
  • the pH of the test and control samples will be adjusted to, and maintained at, a pH which approximates neutrality (i.e. pH 6.5-8.0) . Such a pH adjustment will prevent complex formation, thereby providing a more accurate quantitation of the level of marker in the sample.
  • the pH of the sample is adjusted and the sample is concentrated in order to enhance the detection of the marker.
  • the level (s) of preeclampsia and preterm birth marker (s) in the biological sample from an individual are evaluated.
  • the level of one or more preeclampsia and preterm birth markers in the subject sample may be evaluated by any convenient method.
  • preeclampsia/preterm birth 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.
  • the level of at least one preeclampsia and preterm birth marker may be evaluated by detecting in a sample the amount or level of one or more proteins/polypeptides or fragments thereof to arrive at a protein level representation.
  • protein and “polypeptide” as used in this application are interchangeable.
  • Polypeptide refers to a polymer of amino acids (amino acid sequence) and does not refer to a specific length of the molecule. Thus peptides and oligopeptides are included within the definition of polypeptide.
  • This term also refers to or includes post-translationally modified polypeptides, for example, glycosylated polypeptide, acetylated polypeptide, phosphorylated polypeptide and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • 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) /Tweenor 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) . Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material.
  • 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 substrate.
  • a urease or peroxidase-conjugated anti-human IgG may be employed, for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hrs at room temperature in a PBS-containing solution such as PBS/Tween) .
  • 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 specificity 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 particle, 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, particle, 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 include but are not limited to mass spectrometry, proteomic arrays, xMAP TM microsphere technology, flow cytometry, western blotting, and immunohistochemistry.
  • the level of at least one preeclampsia and preterm birth marker may be evaluated by detecting 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 detected using any convenient protocol. While a variety of different manners of detecting nucleic acids 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" nucleic acids for each of the genes to be assayed/profiled in the marker representation to be generated is employed.
  • a sample of target nucleic acids is first prepared from the initial nucleic acid sample being assayed, where preparation may include labeling of the target nucleic acids with a label, e.g., a member of signal producing system.
  • a label e.g., a member of signal producing system.
  • the sample is contacted with the array under hybridization conditions, whereby complexes are formed between target nucleic acids that are complementary to probe sequences attached to the array surface. The presence of hybridized complexes is then detected, either qualitatively or quantitatively.
  • an array of "probe" nucleic acids that includes a probe for each of the phenotype determinative genes whose expression is being assayed is contacted with target nucleic acids as described above. Contact is carried out under hybridization conditions, e.g., stringent hybridization conditions, and unbound nucleic acid is then removed.
  • hybridization conditions e.g., stringent hybridization conditions
  • unbound nucleic acid is then removed.
  • stringent assay conditions refers to conditions that are compatible to produce binding pairs of nucleic acids, e.g., surface bound and solution phase nucleic acids, of sufficient complementarity to provide for the desired level of specificity in the assay while being less compatible to the formation of binding pairs between binding members of insufficient complementarity to provide for the desired specificity. Stringent assay conditions are the summation or combination (totality) of both hybridization and wash conditions.
  • the resultant pattern of hybridized nucleic acid provides information regarding expression for each of the genes that have been probed, where the expression information is in terms of whether or not the gene is expressed and, typically, at what level, where the expression data, i.e., marker representation (e.g., in the form of a transcriptosome) , may be both qualitative and quantitative.
  • non-array based methods for quantitating the level of one or more nucleic acids in a sample may be employed, including those based on amplification protocols, e.g., Polymerase Chain Reaction (PCR) -based assays, including quantitative PCR, reverse-transcription PCR (RT-PCR) , real-time PCR, and the like.
  • PCR Polymerase Chain Reaction
  • RT-PCR reverse-transcription PCR
  • real-time PCR real-time PCR
  • 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 acid 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 concentration (s) 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 level of each of the panel of markers is fed, by for example inputting to a computer, into the random forest model to provide the preeclampsia assessment and prognose preterm birth.
  • the preeclampsia assessment and preterm birth prognosing may be employed to diagnose a preeclampsia and predict preterm birth; that is, to provide a determination as to whether a subject is affected by preeclampsia or will be affected by preterm birth, the type of preeclampsia and/or preterm birth, the severity of preeclampsia and/or preterm birth, etc.
  • the subject may present with clinical symptoms of preeclampsia, e.g. elevated blood pressure (e.g.
  • 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 present with risk factors of preterm birth, e.g. diabetes, high blood pressure, being pregnant with more than one baby, being either obese or underweight, a number of vaginal infections, tobacco smoking, and psychological stress, among others.
  • the subject may be asymptomatic for preeclampsia/preterm birth and have no risk factors associated with preeclampsia/preterm birth.
  • the preeclampsia assessment and preterm birth prognosing may be employed to prognose a preeclampsia and/or preterm birth; that is, to provide a preeclampsia and/or preterm birth prognosis.
  • the preeclampsia and preterm birth marker level representation may be used to predict a subject's susceptibility, or risk, of developing preeclampsia and preterm birth.
  • preeclampsia and preterm birth marker level representation may be used to predict the course of disease progression and/or disease outcome, e.g. expected onset of the preeclampsia and/or preterm birth, expected duration of the preeclampsia, expectations as to whether the preeclampsia will develop into eclampsia, etc.
  • the preeclampsia and preterm birth marker level representation may be used to predict a subject's responsiveness to treatment for the preeclampsia and preterm birth, e.g., positive response, a negative response, no response at all.
  • the preeclampsia assessment and preterm birth prognosing may be employed to monitor a preeclampsia or preterm birth.
  • monitoring e.g. to inform a preeclampsia/preterm birth diagnosis, to inform a preeclampsia/preterm birth prognosis, to provide information as to the effect or efficacy of a preeclampsia/preterm birth treatment, and the like.
  • the preeclampsia assessment and preterm birth prognosing may be employed to determine a treatment for a subject.
  • treatment , “treating” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, and includes: (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.
  • Preeclampsia 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/preterm birth assessment may comprise additional assessment (s) that are employed in conjunction with the methods stated above.
  • the subject methods may further comprise measuring one or more clinical parameters/factors associated with preeclampsia, e.g. blood pressure, urine protein, weight changes, water retention (edema) , liver enzyme levels, and platelet count.
  • a subject maybe assessed for one or more clinical symptoms, e.g.
  • a positive outcome of the clinical assessment i.e. the detection of one or more symptoms associated with preeclampsia/preterm birth
  • a positive outcome of the clinical assessment i.e. the detection of one or more symptoms associated with preeclampsia/preterm birth
  • the marker level representation to provide a preeclampsia/preterm birth diagnosis, a preeclampsia/preterm birth prognosis, to monitor the preeclampsia/preterm birth, etc.
  • the clinical parameters may be measured prior to obtaining the preeclampsia and preterm birth marker level representation, for example, to inform the artisan as to whether a preeclampsia and preterm birth marker level representation should be obtained, e.g. to make or confirm a preeclampsia/preterm birth diagnosis.
  • the clinical parameters may be measured after obtaining the preeclampsia and preterm birth marker level representation, e.g. to monitor a preeclampsia/preterm birth.
  • the subject methods of providing a preeclampsia assessment and prognosing preterm birth may further comprise assessing one or more factors associated with the risk of developing preeclampsia/preterm birth.
  • preeclampsia/preterm birth risk factors include, for example, a medical condition such as gestational diabetes, obesity, chronic hypertension, renal disease, a thrombophilia; age of greater than 35 years or less than 20 years; a family history of preeclampsia; nulliparity; preeclampsia in a previous pregnancy; stress; being pregnant with more than one baby; being either obese or underweight; a number of vaginal infections; tobacco smoking.
  • a subject maybe assessed for one or more risk factors, e.g. medical condition, family history, etc., when pregnancy is first confirmed or thereafter, wherein a positive outcome of the risk assessment (i.e. the determination of one or more risk factors associated with preeclampsia/preterm birth) is used in combination with the marker level representation to provide a preeclampsia/preterm birth diagnosis, a preeclampsia/preterm birth prognosis, to monitor the preeclampsia/preterm birth, etc.
  • a positive outcome of the risk assessment i.e. the determination of one or more risk factors associated with preeclampsia/preterm birth
  • the marker level representation i.e. the determination of one or more risk factors associated with preeclampsia/preterm birth
  • 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 and prognosing preterm birth include providing a diagnosis, prognosis, or result of the monitoring.
  • the preeclampsia/preterm birth 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/preterm birth, of the type, stage, or severity of the subject's preeclampsia/preterm birth, etc.
  • preeclampsia/preterm birth diagnosis a "preeclampsia/preterm birth diagnosis" ; the artisan's prediction of the patient's susceptibility to developing preeclampsia/preterm birth, of the course of disease progression, of the patient's responsiveness to treatment, etc. (i.e., the artisan's "preeclampsia/preterm birth prognosis” ) ; or the results of the artisan's monitoring of the preeclampsia/preterm birth.
  • 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 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) .
  • 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 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.
  • 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 and preterm birth marker representation, e.g. a preeclampsia/preterm birth profile or a preeclampsia/preterm birth score, as discussed in greater detail above.
  • a subject report includes at least an artisan's preeclampsia/preterm birth assessment, e.g.
  • 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/preterm birth patients versus healthy individuals, i.e. individuals that do not have and/or do not develop preeclampsia/preterm birth.
  • 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 information about the service provider, which may be located outside the healthcare facility at which the user is located, or within the healthcare facility. Examples of such information can include the name and location of the service provider, the name of the reviewer, and where necessary or desired the name of the individual who conducted sample gathering and/or data generation. Report fields with this information can generally be populated using data entered by the user, which can be selected from among pre-scripted selections (e.g., using a drop-down menu) . Other service provider information in the report can include contact information for technical information about the result and/or about the interpretive report.
  • the report may include a patient data section, including patient medical history (which can include, e.g., age, race, serotype, prior preeclampsia/preterm birth 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 physician, 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/preterm birth 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 (DO
  • the report may include a sample data section, which may provide information about the biological sample analyzed in the monitoring assessment, such as the source of biological sample obtained from the patient (e.g. blood, saliva, or type of tissue, etc. ) , how the sample was handled (e.g. storage temperature, preparatory protocols) and the date and time collected. Report fields with this information can generally be populated using data entered by the user, some of which may be provided as pre-scripted selections (e.g., using a drop-down menu) .
  • the report may include a results section.
  • the report may include a section reporting the results of a protein level determination assay (e.g., "6.0 ng/ml Activin A in serum” ) , or a calculated preeclampsia/preterm birth score.
  • 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/preterm birth.
  • the interpretive report can include a diagnosis of preeclampsia/preterm birth.
  • the interpretive report can include a characterization of preeclampsia/preterm birth.
  • the assessment portion of the report can optionally also include a recommendation (s) . For example, where the results indicate that preeclampsia/preterm birth 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.
  • the report can include all or some of the elements above, with the proviso that the report generally includes at least the elements sufficient to provide the analysis requested by the user (e.g. a calculated preeclampsia and preterm birth marker level representation; a prediction, diagnosis or characterization of preeclampsia/preterm birth) .
  • 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 determining the level of preeclampsia and preterm birth 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 and preterm birth marker, for example, 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 and preterm birth marker simultaneously,
  • One type of reagent that is specifically tailored for determining the level is a collection of antibodies that bind specifically to the protein markers, e.g. in an ELISA format, in an xMAP TM microsphere format, on a proteomic array, in suspension for analysis by flow cytometry, by western blotting, by dot blotting, or by immunohistochemistry. Methods for using the same are well understood in the art. These antibodies can be provided in solution. Alternatively, they may be provided pre-bound to a solid matrix, for example, the wells of a multi-well dish or the surfaces of xMAP microspheres.
  • 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.
  • Another type of reagent that is specifically tailored for determining the level of genes is a collection of gene specific primers that is designed to selectively amplify such genes (e.g., using a PCR-based technique, e.g., real-time RT-PCR) .
  • Gene specific primers and methods for using the same are described in U.S. Patent No. 5,994,076, the disclosure of which is herein incorporated by reference.
  • 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 selected from the group consisting of Activin A and Adam12, or a biochemical substrate specific for the cofactor/prosthetic group heme, in some instances for a plurality of these genes/polypeptides, e.g., at least 2, 3, 4, 5, 6, 7, 8 or more genes/polypeptides.
  • the collection of probes, primers, or antibodies includes reagents specific for Activin A and Adam12 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/cofactors that are listed above, or they may include reagents specific for additional genes/proteins/cofactors that are not listed above, such as probes, primers, or antibodies specific for genes/proteins/cofactors whose expression pattern are known in the art to be associated with preeclampsia/preterm birth.
  • 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 acid or protein, respectively may be coupled with an electrochemical biosensor platform that will allow multiplex determination of these biomarkers for personalized preeclampsia/preterm birth care.
  • the systems and kits of the subject invention may include the above-described arrays, gene-specific primer collections, or protein-specific antibody collections.
  • the systems and kits may further include one or more additional reagents employed in the various methods, such as primers for generating target nucleic acids, dNTPs and/or rNTPs, which may be either premixed or separate, one or more uniquely labeled dNTPs and/or rNTPs, such as biotinylated or Cy3 or Cy5 tagged dNTPs, gold or silver particles with different scattering spectra, or other post synthesis labeling reagent, such as chemically active derivatives of fluorescent dyes, enzymes, such as reverse transcriptases, DNA polymerases, RNA polymerases, and the like, various buffer mediums, e.g.
  • 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 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.
  • preeclampsia is a pregnancy-related vascular disorder affecting 5%-8%of all pregnancies (Berg et al. Overview of maternal morbidity during hospitalization for labor and delivery in the United States: 1993-1997 and 2001-2005. Obstetrics and gynecology 2009; 113: 1075-81; Mackay et al. Pregnancy-related mortality from preeclampsia and eclampsia. Obstetrics and gynecology 2001; 97: 533-8) .
  • PE which often causes fetal growth restriction and pre-term delivery as well as fetal mortality and morbidity, can be remedied by delivery of the placenta and fetus (Powe et al. Preeclampsia, a disease of the maternal endothelium: the role of antiangiogenic factors and implications for later cardiovascular disease. Circulation 2011; 123: 2856-69) .
  • the etiology of PE is incompletely understood. Current diagnosis of PE is based on the signs of hypertension and proteinuria (Gynecologists ACOOA ACOG practice bulletin. Diagnosis and management of preeclampsia and eclampsia. Number 33, January 2002.
  • PE is a multisystem disorder of pregnancy with the placenta playing a pivotal role.
  • Investigators have used genetic, genomic and proteomic 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. Placenta 2007; 28: 487-97; Loset et al.
  • Dynamic proteome in enigmatic preeclampsia an account of molecular mechanisms and biomarker discovery. Proteomics Clinical applications 2012; 6: 79-90; Carty et al. Urinary proteomics for prediction of preeclampsia. Hypertension 2011; 57: 561-9) have also revealed candidate biomarkers for future testing. 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.
  • sFlt-1 elevated soluble fms-like tyrosine kinase
  • PIGF placental growth factor
  • 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.
  • Excess placental soluble fms-like tyrosine kinase 1 (sFIt-1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.
  • Circulating levels of the antiangiogenic marker sFLT-1 are increased in first versus second pregnancies.
  • PBMCs peripheral blood mononuclear cells
  • PTB preterm birth
  • Risk factors include diabetes, high blood pressure, being pregnant with more than one baby, being either obese or underweight, a number of vaginal infections, tobacco smoking, and psychological stress, among others. It is recommended that labor not be medically induced before 39 weeks unless required for other medical reasons. The same recommendation applies to cesarean section. Preterm labor and delivery continue to plague modern obstetrics. The preterm birth rate still rests at approximately 11%of deliveries, with ensuing neonatal morbidity and death. This is unchanged despite research into strategies such as tocolytics, risk assessment, and regionalization.
  • Neonatal survival has improved by advances in the neonatal intensive care unit and the use of antepartum steroid administration to reduce the incidence of outcomes (such as respiratory distress syndrome and intraventricular hemorrhage) .
  • outcomes such as respiratory distress syndrome and intraventricular hemorrhage
  • the use of different markers most notably the presence of bacterial vaginosis, assessment of cervicovaginal fetal fibronectin, and cervical length determined by ultrasound scanning, has been studied in the hopes of targeting those women who are at risk for premature delivery, thereby aiding the clinician in decision making to treat specific patients with different modalities (eg, tocolytics, steroids, antibiotics, cerclage) .
  • a serum molecular marker would be advantageous because cervical length, fetal fibronectin, and bacterial vaginosis status involve cervical/vaginal evaluation.
  • 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/PTB diagnosis.
  • PE antiphospholipid syndrome
  • SLE systemic lupus erythematosus
  • preterm laboring symptoms regular uterine contractions, low abdominal cramping, low back pain, pelvic pressure, vaginal bleeding, and increased vaginal discharge
  • IUGR intrauterine growth retardation
  • HELLP syndrome HELLP syndrome
  • Placenta 2013; 36: 160-169) and three placenta expression studies were combined and subjected to multiplex meta-analysis with the method we previously developed (Morgan et al. Comparison of multiplex meta-analysis techniques for understanding the acute rejection of solid organ transplants. BMC bioinformatics 2010; 11 Suppl 9: S6; Chen et al. Differentially expressed RNA from public microarray data identifies serum protein biomarkers for cross-organ transplant rejection and other conditions. PLoS computational biology 2010; 6) . For each of the 22, 394 genes tested, we calculated the meta-fold change across all studies. Significant genes were selected if they were measured in 5 or more studies and the meta effect p value was less than 0.05 and the meta-fold change higher than 1.2.
  • ROC curve analysis Zweig et al. Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clinical chemistry 1993; 39: 561-77; Sing et al. ROCR: visualizing classifier performance in R. Bioinformatics 2005; 21: 3940-1) .
  • a composite panel combining all significant biomarkers was developed using random forest algorithm. The biomarker panel score was defined as the majority votes of all trees in random forest algorithm.
  • Previous placental expression studies were combined for a multiplex meta-analysis, as well as Protein Atlas analysis and human orthologous gene analysis, to discover biomarker candidates diagnosing PE and PTB from normal controls. This effort identified Activin A and Adam12 as differential placental biomarkers for both PE and PTB from normal controls.
  • BMI body mass index
  • BP blood pressure
  • whisker box and scatter plots in Figures 7-8 two proteins were validated by ELISA assays (Mann–Whitney U-test) .
  • Figures 7-8 also demonstrated the distribution of maternal serum abundance of each validated protein over the gestational age (weeks) of blood sample collection, delivery, and the gap in between.
  • Table 9 and 10 are summarized in Table 9 and 10.
  • Forest plots summarize the PE to control ratios of 21 PE markers and PTB to control ratios of 32 PTB markers across placental expression meta-analyses.
  • the biomarkers derived from the proteomic and expression meta-analyses consistently shared the same trend of up-or down-regulation between PE, PTB, and control samples.
  • Biomarker panel construction Using data from the ELISA assays, we constructed a random forest algorithm of the two protein analyte panel (Activin A and Adam12) , plus clinical parameters ( Figures 9-11) . We sought to identify biomarker panels of optimal feature number, balancing the need for small panel size, accuracy of classification, goodness of class separation (case versus control) , and sufficient sensitivity and specificity. Figures 9-11 shows that all the panels having 100%sensitivity and 100%specificity.
  • Pathway analysis of biomarkers We analyzed the validated biomarkers that are significantly differentially expressed in preterm birth as a composite, using PathVisio software (version 3.2.1, an open-source pathway analysis and drawing software) (Martijn et al. Presenting and exploring biological pathways with PathVisio. BMC Bioinformatics 2008; 9 (1) : 399) .
  • PathVisio software version 3.2.1, an open-source pathway analysis and drawing software
  • our pathway analysis led to the identification of the following statistically significant canonical pathways which may play important roles in preterm birth pathophysiology:
  • Activin A is a homodimeric protein that consists of 2 ⁇ A subunits.
  • Activin A is a member of the transforming growth factor- ⁇ family and is related to inhibin A. It has pleotropic actions, including the stimulation of follicle-stimulating hormone release in the anterior pituitary, a role in neuronal health, and an effect on body axis development; it is produced in a variety of tissues, such as brain, pituitary gland, gonads, bone marrow, and placenta; evidence that supports an Activin A role in pregnancy results from both in vitro and clinical studies. All of these support our hypothesis that preterm birth is associated with increased shedding of placental debris leading to increased plasma levels of biomarker proteins, which could contribute to the inflammatory response, hormone imbalance, and endothelial dysfunction.
  • the protein levels of a panel of preeclampsia markers was statistically assessed to determine how to weigh the contribution of each polypeptide to a preeclampsia and preterm birth score for a sample based on this panel.
  • Adam12 levels were determined to be least significant; and Activin A levels were determined to be most significant, i.e. about 11.6-fold more significant than Adam12.
  • Table 1 The microarray datasets of preeclampsia studied in present work.
  • Table 4 Clinical information of the enrolled case and control subjects. Clinical information was unavailable for one control subject. a. Kruskal-Wallis test; b. Chi-squared test.

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Abstract

L'invention concerne un procédé destiné à l'estimation de pré-éclampsie et au pronostic de naissance prématurée chez un sujet, qui consiste : à développer et à entraîner un modèle de forêt aléatoire pour déduire une mesure de notation pour différencier un sujet normal, un sujet à pré-éclampsie et un sujet à naissance prématurée; à évaluer un ensemble de marqueurs du sujet pour déterminer le niveau de chaque marqueur de l'ensemble de marqueurs; et à injecter le niveau de chaque marqueur de l'ensemble de marqueurs dans le modèle de forêt aléatoire pour procéder à l'estimation de pré-éclampsie et pronostiquer une naissance prématurée. L'ensemble de marqueurs comprend l'inhibine bêta A (activine A) et au moins un marqueur sélectionné parmi un groupe constitué d'ADAM métallopeptidase domaine 12 (ADAM12), de l'indice de masse corporelle (IMC) et du nombre de globules blancs (WBC).
PCT/CN2016/103060 2016-10-24 2016-10-24 Procédés et trousses destinés à l'estimation de pré-éclampsie et au pronostic de naissance prématurée WO2018076134A1 (fr)

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CN112126683A (zh) * 2020-08-31 2020-12-25 中山大学 用于迟发型子痫前期诊断的标志物
CN112831522B (zh) * 2021-02-18 2022-09-16 中国人民解放军陆军特色医学中心 一种条件性敲入人Siglec-6分子的小鼠模型及其构建方法

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CN112180098B (zh) * 2019-12-06 2023-02-17 中山大学 胎盘相关疾病标志物的筛选方法及标志物

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