WO2018223008A1

WO2018223008A1 - Predictive factors for predicting laparotomy closure

Info

Publication number: WO2018223008A1
Application number: PCT/US2018/035620
Authority: WO
Inventors: Rondi GELBARD; Seth A. SCHOBEL-MCHUGH; Eric A. ELSTER; Beverly J. GAUCHER
Original assignee: Henry M Jackson Foundation for Advancedment of Military Medicine Inc
Current assignee: Henry M Jackson Foundation for Advancedment of Military Medicine Inc
Priority date: 2017-06-02
Filing date: 2018-06-01
Publication date: 2018-12-06
Anticipated expiration: 2019-12-02

Abstract

The present invention relates to methods of determining if a subject has an increased risk of developing unsuccessful fascial closure after laparotomy (UFC) prior to the onset of any detectable symptoms thereof. The methods comprise analyzing at least one sample from the subject to determine a value of the subject's risk profile and comparing the value of the subject's risk profile with the value of a normal risk profile. A change in the value of the subject's risk profile, over or under normal values is indicative that the subject has an increased risk of having or developing symptoms associated with UFC prior to the onset of any detectable symptoms thereof.

Description

PREDICTIVE FACTORS FOR PREDICTING LAPAROTOMY CLOSURE

Statement Regarding Federally Sponsored Research or Development

[0001] This invention was made with government support under HT9404-13-1 and H U0001-15-2- 0001 awarded by The Department of Defense. The government has certain rights in the invention.

Background of the Invention

Field of the Invention

[0002] The present invention relates to methods of determining if a su bject has an increased risk of unsuccessful fascial closure after laparotomy (U FC) prior to the onset of any detectable symptoms thereof. The methods comprise analyzing at least one sample from the subject to determine a value of the subject's risk profile and comparing the value of the subject's risk profile with the value of a normal risk profile. A change in the value of the subject's risk profile, over or under normal values is indicative that the subject has an increased risk of having or developing symptoms associated with U FC prior to the onset of any detectable symptoms thereof.

Background of the Invention

[0003] The time of closure of combat-related extremity wounds is planned based on the extent of injury and assessment of local signs of healing such as the absence of tissue necrosis and resolution of infection after multiple surgical debridements (SD). Approximately 80% of these wounds are successfully closed without subsequent complications such as wound failure. Successful wound healing is associated with a distinct local and systemic cytokine response. The present disclosure investigates if unsuccessful fascial closure after laparotomy (U FC) could be estimated based on the cytokine and procalcitonin (ProCT) levels .

Summary of the Invention

[0004] The present invention relates to methods of determining if a su bject has an increased risk of unsuccessful fascial closure after laparotomy (U FC) prior to the onset of any detectable symptoms thereof. The methods comprise analyzing at least one sample from the subject to determine a value of the subject's risk profile and comparing the value of the subject's risk profile with the value of a normal risk profile. A change in the value of the subject's risk profile, over or under normal values is indicative that the subject has an increased risk of having or developing symptoms associated with UFC prior to the onset of any detectable symptoms thereof.

[0005] The present invention encompasses a method of treating unsuccessful fascial closure after laparotomy in a human subject comprising (a) obtaining a first blood, serum or plasma sample from the human subject, (b) determining levels of one or more polypeptides selected from the group consisting of procalcitonin (ProCT) and lnterleukin-4 (I L-4) in the first blood, serum or plasma sample, and (c) surgically closing the laparotomy within 24 hours in subjects with polypeptide levels about the same as human subjects with successful facial closure after laparotomy. In some embodiments, the method further comprises (d) obtaining a second blood, serum or plasma sample from the human subject at least 24 hours after obtaining the first blood, serum or plasma sample, and (e) determining levels of one or more polypeptides selected from ProCT and IL-4 in the second blood, serum or plasma sample.

[0006] In some embodiments, the method further comprises obtaining a first peritoneal lavage sample from the human subject, and determining levels of one or more polypeptides selected from ProCT and I L-17 in the first peritoneal lavage sample. In some embodiments, the method further comprises obtaining a second blood, serum or plasma sample from the human subject, obtaining a second peritoneal lavage sample from the human subject, determining levels of one or more polypeptides selected from ProCT and I L-4 in the second blood, serum or plasma sample, determining levels of one or more polypeptides selected from ProCT and I L-17 in the second peritoneal lavage sample.

[0007] The present invention encompasses a method of treating unsuccessful fascial closure after laparotomy in a human subject comprising (a) obtaining a first peritoneal lavage sample from the human subject, (b) determining levels of one or more polypeptides selected from ProCT and lnterleukin-17 (I L-17) in the first peritoneal lavage sample, and (c) surgically closing the laparotomy within 24 hours in subjects with polypeptide levels about the same as human subjects with successful fascial closure after laparotomy. In some embodiments, the invention further comprises (d) obtaining a second peritoneal lavage sample from the human subject at least 24 hours after obtaining the first peritoneal lavage sample, and (e) determining levels of one or more polypeptides selected from ProCT and I L-17 in the second peritoneal lavage sample.

[0008] In some embodiments, the second sample is obtained 48 to 72 hours after obtaining the first sample. In some embodiments, the laparotomy is surgically closed after determining polypeptide levels in the second sample. In some embodiments, the human subject has a trauma wound. In some embodiments, the trauma wound is an abdominal wound.

[0009] The invention also encompasses a method of determining whether to surgically close a laparotomy in a human subject comprising (a) obtaining a blood, serum or plasma sample from the human subject; and (b) measuring levels of one or more polypeptides selected from ProCT and I L-4 in the blood, serum or plasma sample; wherein polypeptide levels about the same as human subjects with successful fascial closure after laparotomy indicate that the wound should be closed within 24 hours.

[0010] The invention encompasses a method of determining whether to surgically close a laparotomy in a human subject comprising: (a) obtaining a peritoneal lavage sample from the human subject; and (b) measuring the levels of ProCT and I L-17 in the peritoneal lavage sample; wherein polypeptide levels about the same as human subjects with successful fascial closure after laparotomy indicate that the laparotomy should be closed within 24 hours. The method may optionally further comprise (c) obtaining a peritoneal lavage sample from the human subject; and (d) measuring the levels of ProCT and I L-17 in the peritoneal lavage sample.

Brief Description of the Drawings

[0011] Figure 1 depicts Receiver Operator Characteristic Curve (ROC) plots for: (A) failed fascial closure, (B) open abdomens, and (C) healed fascial closure.

[0012] Figure 2 depicts a box and whisker plot of the distribution of procalcitonin levels as measured from serum of patients with a variety of closure outcomes (healed, failed, open).

[0013] Figure 3 depicts a box and whisker plot of the distribution of procalcitonin levels as measured from peritoneal lavage fluid of patients with a variety of closure outcomes (healed, failed, open).

Detailed Description of the Invention

[0014] The present invention relates to methods of determining if a su bject has an increased risk of unsuccessful fascial closure after laparotomy (U FC) prior to the onset of any detectable symptoms thereof. The methods comprise analyzing at least one sample from the subject to determine a value of the subject's risk profile and comparing the value of the subject's risk profile with the value of a normal risk profile. A change in the value of the subject's risk profile, over or under normal values is indicative that the subject has an increased risk of having or developing symptoms associated with U FC prior to the onset of any detectable symptoms thereof.

[0015] The present invention also relates to methods of detecting elevated levels of a specific collection of analytes in one or more samples obtained from a subject. In one embodiment, the collection of analytes comprises serum levels of procalcitonin (ProCT) and interleukin-4 ( IL-4), and peritoneal lavage levels of ProCT and interleukin-17 (I L-17). [0016] As used herein, the term "subject" or "test subject" indicates a mammal, in particular a human or non-human primate. The test subject is in need of an assessment of susceptibility of U FC. For example, the test subject may have no symptoms that U FC may occur.

[0017] The term unsuccessful fascial closure after laparotomy, or U FC, is used herein to mean the inability to bring the muscles of the abdominal wall together primarily, or dehiscence of the abdominal wall after initial closure without the ability to bring the muscles together again.

[0018] As used herein, the term means "increased risk" is used to mean that the test subject has an increased chance of developing U FC compared to a normal individual. The increased risk may be relative or absolute and may be expressed qualitatively or quantitatively. For example, an increased risk may be expressed as simply determining the subject's risk profile and placing the patient in an "increased risk" category, based upon previous population studies. Alternatively, a numerical expression of the subject's increased risk may be determined based upon the risk profile. As used herein, examples of expressions of an increased risk include but are not limited to, odds, probability, odds ratio, p-values, attributable risk, biomarker index score, relative frequency, positive predictive value, negative predictive value, and relative risk.

[0019] For example, the correlation between a subject's risk profile and the likelihood of developing UFC may be measured by an odds ratio (OR) and by the relative risk (RR). If P(R⁺) is the probability of developing U FC for individuals with the risk profile (R) and P( R^") is the probability of developing U FC for individuals without the risk profile, then the relative risk is the ratio of the two probabilities: RR=P(R⁺)/P(R-).

[0020] In case-control studies, however, direct measures of the relative risk often cannot be obtained because of sampling design. The odds ratio allows for an approximation of the relative risk for low-incidence diseases and can be calculated: OR=(F⁺/( l-F⁺))/(F^"/(l-F^")), where F⁺ is the frequency of a risk profile in cases studies and F^" is the frequency of risk profile in controls. F⁺ and F^" can be calculated using the risk profile frequencies of the study.

[0021] The attributable risk (AR) can a lso be used to express an increased risk. The AR describes the proportion of individuals in a population exhibiting UFC to a specific member of the risk profile. AR may also be important in quantifying the role of individual components (specific member) in condition etiology and in terms of the public health impact of the individual risk factor. The public health relevance of the AR measurement lies in estimating the proportion of cases of U FC in a population of wounded subjects that could be prevented if the profile or individual factor were absent. AR may be determined as follows: AR=P_E(RR-1)/(PE(RR-1)+1), where AR is the risk attributable to a profile or individual factor of the profile, and PE is the frequency of exposure to a profile or individual component of the profile within the population at large. RR is the relative risk, which can be approximated with the odds ratio when the profile or individual factor of the profile under study has a relatively low incidence in the general population.

[0022] In one embodiment, the increased risk of a patient can be determined from p-values that are derived from association studies. Specifically, associations with specific profiles can be performed using regression analysis by regressing the risk profile with the presence or absence of UFC. In addition, the regression may or may not be corrected or adjusted for one or more factors. The factors for which the analyses may be adjusted include, but are not limited to age, sex, weight, ethnicity, type of wound, number of wounds, number of days from injury, number of previous surgical debridements, geographic location, fasting state, state of pregnancy or post-pregnancy, menstrual cycle, general health of the subject, alcohol or drug consumption, caffeine or nicotine intake and circadian rhythms, to name a few.

[0023] Increased risk can also be determined from p-values that are derived using logistic regression. Binomial (or binary) logistic regression is a form of regression which is used when the dependent is a dichotomy and the independents are of any type. Logistic regression can be used to predict a dependent variable on the basis of continuous or categorical or both (continuous and categorical) independents and to determine the percent of variance in the dependent variable explained by the independents; to rank the relative importance of independents; to assess interaction effects; and to understand the impact of covariate control variables. Logistic regression applies maximum likelihood estimation after transforming the dependent into a "logit" variable (the natural log of the odds of the dependent occurring or not). In this way, logistic regression estimates the probability of a certain event occurring. These analyses may be conducted with virtually any statistics program, such as not limited to SAS, R package available through CRAN repository.

[0024] SAS ("statistical analysis software") is a general purpose package (similar to Stata and SPSS) created by Jim Goodnight and N.C. State University colleagues. Ready-to-use procedures handle a wide range of statistical analyses, including but not limited to, analysis of variance, regression, categorical data analysis, multivariate analysis, survival analysis, psychometric analysis, cluster analysis, and nonparametric analysis. R package is free, general purpose package that complies with and runs on a variety of UNIX platforms.

[0025] Accordingly, select embodiments of the present invention comprise the use of a computer comprising a processor and the computer is configured or programmed to generate one or more risk profiles and/or to determine statistical risk. The methods may also comprise displaying the one or risk profiles on a screen that is communicatively connected to the computer. In another embodiment, two different computers can be used: one computer configured or programmed to generate one or more risk profiles and a second computer configured or programmed to determine statistical risk. Each of these separate computers can be communicatively linked to its own display or to the same display.

[0026] As used herein, the phrase "risk profile" means the combination of a subject's risk factors analyzed or observed. The terms "factor" and/or "component" are used to mean the individual constituents that are assessed when generating the profile. The risk profile is a collection of measurements, such as but not limited to a quantity or concentration, for individual factors taken from a test sample of the subject. Examples of test samples or sources of components for the risk profile include, but are not limited to, biological fluids, which can be tested by the methods of the present invention described herein, and include but are not limited to whole blood, such as but not limited to peripheral blood, serum, plasma, cerebrospinal fluid, wound effluent, peritoneal lavage collection, urine, amniotic fluid, lymph fluids, various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, white blood cells, myelomas and the like.

[0027] The risk profile can include a "biological effector" aspect and/or a non-biological effector aspect. As used herein, the term "biological effector" is used to mean a molecule, such as but not limited to, a protein, peptide, a carbohydrate, a fatty acid, a nucleic acid, a glycoprotein, a proteoglycan, etc. that can be assayed. Specific examples of biological effectors can include, cytokines, growth factors, antibodies, hormones, cell surface receptors, cell surface proteins, carbohydrates, etc. More specific examples of biological effectors include interleukins (I Ls) such as I L-la, IL-Ιβ, I L-1 receptor antagonist (I L-1RA), I L-2, I L-2 receptor (I L-2R), I L-3, I L-4, I L-5, I L-6, I L-7, I L-8, I L-10, I L-12, I L-13, I L-15, I L-17, as well as growth factors such as tumor necrosis factor alpha (TN Fa), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), interferon alpha (I N F-a), interferon gamma (I FN-y), epithelial growth factor (EGF), basic endothelial growth factor (bEGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and chemokines such as monocyte chemoattractant protein-1 (CCL2/MCP-1), macrophage inflammatory protein-1 alpha (CCL3/M I P-la), macrophage inflammatory protein-1 beta (Ο- /Μ Ι Ρ-Ιβ), CCL5/RANTES, CCLll/eotaxin, monokine induced by gamma interferon (CXCL9/MIG) and interferon gamma-induced protein-10 (CXCL10/I P10).

[0028] Techniques to assay levels of individual components of the risk profile from test samples are well known to the skilled technician, and the invention is not limited by the means by which the components are assessed. In one embodiment, levels of the individual factors in the serum or wound effluent of the risk profile are assessed using mass spectrometry in conjunction with ultra- performance liquid chromatography (U PLC), high-performance liquid chromatography (H PLC), gas chromatography (GC), gas chromatography/mass spectroscopy (GC/MS), and UPLC to name a few. Other methods of assessing levels of some of the individual components include biological methods, such as but not limited to ELISA assays, Western Blot and multiplexed immunoassays etc. Other techniques may include using quantitative arrays, PCR, Northern Blot analysis. To determine levels of components or factors, it is not necessary that an entire component, e.g., a full length protein or an entire RNA transcript, be present or fully sequenced. In other words, determining levels of, for example, a fragment of protein being analyzed may be sufficient to conclude or assess that an individual component of the risk profile being analyzed is increased or decreased. Similarly, if, for example, arrays or blots are used to determine component levels, the presence/absence/strength of a detectable signal may be sufficient to assess levels of components.

[0029] As used herein, the term non-biological effector is a component that is generally considered not to be a specific molecule. Although not a specific molecule, a non-biological effector may nonetheless still be quantifiable, either through routine measurements or through measurements that stratify the data being assessed. For example, number or concentrate of red blood cells, white blood cells, platelets, coagulation time, blood oxygen content, etc. would be a non-biological effector component of the risk profile. All of these components are measureable or quantifiable using routine methods and equipment. Other non-biological components include data that may not be readily or routinely quantifiable or that may require a practitioner's judgment or opinion. For example, wound severity may be a component of the risk profile. While there may be published guidance on classifying wound severity, stratifying wound severity and, for example, assigning a numerical value to the severity, still involves observation and, to a certain extent, judgment or opinion. In some instances the quantity or measurement assigned to a non-biological effector could be binary, e.g., "0" if absent or "1" if present. In other instances, the non-biological effector aspect of the risk profile may involve qualitative components that cannot or should not be quantified.

[0030] In one embodiment, the mechanism of injury is included in the risk profile. As used herein, the phrase "mechanism of injury" means the manner in which the subject received an injury. For example, the mechanism of injury may be described as a gunshot wound, a vehicle accident, laceration, etc. In another embodiment, data regarding injury type is included in the risk profile. In another embodiment, data on the occurrence of multiple wounds is included in the risk profile. In another embodiment, data on the number of days from injury is included in the risk profile. [0031] Other examples of individual components of the risk profile include but are not limited to ISS score of head, ISS score of chest (thorax) and critical colonization. "ISS score" (injury severity score) is well-known in the art and is used routinely in clinics to assess severity of wounds or injuries.

[0032] As used herein, the term critical colonization (or "CC") is a measure of CFU that the subject has in serum and/or tissue for at least one wound when initially examined by the attending physician. For example, if a subject has CFU of lxlO⁵ per ml of serum, or if at least one wound has CFU of 1x10^s per mg of tissue, the subject is said to be "positive" for CC. If the total serum CFU or no single would has CFU of at least lxlO⁵ the subject is said to be "negative" for CC.

[0033] To determine which of the biological effector or non-biological effector components may be critical in the subjects' risk profiles, routine statistical methods can be employed. For example, rflmpute from the randomForest R package can be used to impute missing data. Up-sampling and predictor rank transformations can be performed on the data set only for variable selection to accommodate class imbalance and non-normality in the data.

[0034] For variable selection, the constraint-based algorithms /asi./amfa, iamb and gs and the constraint-based local discovery learning algorithms mmpc and si.hiton.pc from the "bnlearn" R package can be used to search the input dataset for nodes of Bayesian networks. The nodes can be chosen as the reduced variable sets. Before running the data through variable selection and binary classification algorithms, the variables may or may not randomly re-ordered. The data can be run through the variable selection and binary classification algorithms more than once, for example, 10, 20, 30, 40, 50 or even more times.

[0035] For binary classification and model selection, each variable set can be pulled from the raw data and run in sundry binary classification algorithms using the train function from the R caret package: linear discriminant analysis {Ida), classification and regression trees {cart), k-nearest neighbors {knn), support vector machine {svm), logistic regression {glm), random forest {rf), generalized linear models {glmnet) and naive Bayes {nb). The best variable set and binary classification algorithm combination that first produces the highest kappa and then the highest sensitivity with reasonable specificity can then be chosen.

[0036] The resultant models are then examined using accuracy, no information rate, positive predictive value and negative predictive value. Model performance can be further assessed using the plot. roc command to compute the Receiver Operator Characteristic Curves (ROC) and area under curve (AUC). The dca R command from the Memorial Sloan Kettering Cancer Center website, www.mskcc.org, can be used to compute the Decision Curve Analysis (DCA). [0037] Finally, for univariate analysis, a Wilcoxon rank-sum test can be used used to identify which biomarkers from specific patient groups are were associated with a specific indication.

[0038] The assessment of the levels of the individual components of the risk profile can be expressed as absolute or relative values and may or may not be expressed in relation to another component, a standard an internal standard or another molecule of compound known to be in the sample. If the levels are assessed as relative to a standard or internal standard, the standard may be added to the test sample prior to, during or after sample processing.

[0039] To assess levels of the individual components of the risk profile, a sample may be taken from the subject. The sample may or may not processed prior assaying levels of the components of the risk profile. For example, whole blood may be taken from an individual and the blood sample may be processed, e.g., centrifuged, to isolate plasma or serum from the blood. The sample may or may not be stored, e.g., frozen, prior to processing or analysis.

[0040] In one embodiment, the individua l levels of each of the risk factors are higher than those compared to normal levels. In another embodiment, one, two, three or four of the levels of each of the factor are higher than normal levels.

[0041] The levels of depletion of the factors or components compared to normal levels can vary. In one embodiment, the levels of any one or more of the factors or components is at least 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 higher than normal levels (where, for sake of clarity, a marker a level of "1" would indicate that the component is at the same level in both the subject and normal samples). For the purposes of the present invention, the number of "times" the levels of a factor are higher over normal can be a relative or absolute number of times. In the alternative, the levels of the factors or components may be normalized to a standard and these normalized levels can then be compared to one another to determine if a factor or component is lower, higher or about the same.

[0042] For the purposes of the present invention the risk profile comprises at least one, two, three or four of the factors or components for the prediction of U FC. If one factor or component of the biological effector aspect of the risk profile is used in generating the risk profile for the prediction of UFC, then any one of the listed factors or components can be used to generate the profile. If two factors or components of the biological effector aspect of the risk profile are used in generating the risk profile for the prediction of U FC, any combination of the two listed above can be used. If three factors or components of the biological effector aspect of the risk profile are used in generating the risk profile for the prediction of U FC, any combination of three of the factors or components listed above can be used. Of course all members or components of the biological effector aspect of the risk profile can be used in generating the risk profile for the prediction of UFC.

[0043] The subject's risk profile is compared to the profile that is deemed to be a normal risk profile. To establish the risk profile of a normal individual, an individual or group of individuals may be first assessed to ensure they have no signs, symptoms or diagnostic indicators of UFC. Once established, the risk profile of the individual or group of individuals can then be determined to establish a "normal risk profile." In one embodiment, a normal risk profile can be ascertained from the same subject when the subject is deemed as healthy with no signs, symptoms or diagnostic indicators of UFC. In one embodiment, a risk profile from a "normal subject," e.g., a "normal risk profile," is a subject with a wound but did not exhibit or display U FC. In one specific embodiment, the normal subject has a chest wound, head wound or as extremity (arm, hand, finger(s), leg, foot, toe(s)) wound but did not exhibit UFC.

[0044] In one embodiment, a "normal" risk profile is assessed in the same subject from whom the sample is taken prior to the onset of any signs, symptoms or diagnostic indicators that they may exhibit UFC. That is, the term "normal" with respect to a risk profile can be used to mean the subject's baseline risk profile prior to the onset of any wounds, signs, symptoms or diagnostic indicators of potential U FC. The risk profile can then be reassessed periodically and compared to the subject's baseline risk profile. Thus, the present invention also includes methods of monitoring the progression of U FC in a subject, with the methods comprising determining the subject's risk profile at more than one time point. For example, some embodiments of the methods of the present invention will comprise determining the subject's risk profile at two, three, four, five, six, seven, eight, nine, 10 or even more time points over a period of time, such as a week or more, two weeks or more, three weeks or more, four weeks or more, a month or more, two months or more, three months or more, four months or more, five months or more, six months or more, seven months or more, eight months or more, nine months or more, ten months or more, 11 months or more, a year or more or even two years. The methods of monitoring a subject's risk of developing UFC would also include embodiments in which the subject's risk profile is assessed before and/or during and/or after treatment of UFC. In other words, the present invention also includes methods of monitoring the efficacy of treatment of U FC by assessing the subject's risk profile over the course of the treatment and after the treatment. In specific embodiments, the methods of monitoring the efficacy of treatment of UFC comprise determining the subject's risk profile at at least one, two, three, four, five, six, seven, eight, nine or 10 or more different time points prior to the receipt of treatment for U FC and subsequently determining the subject's risk profile at at least one, two, three, four, five, six, seven, eight, nine or 10 or more different time points after beginning of treatment for UFC, and determining the changes, if any, in the risk profile of the subject. The treatment may be any treatment designed to cure, remove or diminish the likelihood of developing U FC.

[0045] In another embodiment, a normal risk profile is assessed in a sample from a different subject or patient (from the subject being analyzed) and this different subject does not have or is not suspected of developing U FC. In still another embodiment, the normal risk profile is assessed in a population of healthy individuals, the constituents of which display no signs, symptoms or diagnostic indicators that they may have or will develop U FC. Thus, the subject's risk profile can be compared to a normal risk profile generated from a single normal sample or a risk profile generated from more than one normal sample.

[0046] Of course, measurements of the individual components, e.g., concentration, ratio, log ratios etc., of the normal risk profile can fall within a range of values, and values that do not fall within this "normal range" are said to be outside the normal range. These measurements may or may not be converted to a value, number, factor or score as compared to measurements in the "normal range." For example, a measurement for a specific factor or component that is below the normal range, may be assigned a value or -1, -2, -3, etc., depending on the scoring system devised.

[0047] In another embodiment, the measurements of the individual components themselves are used in the risk profile, and these levels can be used to provide a "binary" value to each component, e.g., "elevated" or "not elevated." Each of the binary values can be converted to a number, e.g., "1" or "0," respectively.

[0048] In one embodiment, the "risk profile value" can be a single value, number, factor or score given as an overall collective value to the individual components of the profile. For example, if each component is assigned a value, such as above, the component value may simply be the overall score of each individual or categorical value. For example, if four components of the risk profile for predicting UFC are used and three of those components are assigned values of "+2" and one are assigned values of the risk profile in this example would be +7, with a normal value being, for example, "0." In this manner, the risk profile value could be a useful single number or score, the actual value or magnitude of which could be an indication of the actual risk of developing U FC, e.g., the "more positive" the value, the greater the risk of developing UFC.

[0049] In another embodiment the "risk profile value" can be a series of values, numbers, factors or scores given to the individual components of the overall profile. In another embodiment, the "risk profile value" may be a combination of values, numbers, factors or scores given to individual components of the profile as well as values, numbers, factors or scores collectively given to a group of components, such as a biological effector portion. In another example, the risk profile value may comprise or consist of individual values, number or scores for specific component as well as values, numbers or scores for a group of components.

[0050] In another embodiment individual values from the risk profile and/or the mechanism of injury can be used to develop a single score, such as a "combined risk index," which may utilize weighted scores from the individual component values reduced to a diagnostic number value. The combined risk index may also be generated using non-weighted scores from the individual component values. When the "combined risk index" exceeds a specific threshold level, determined by a range of values developed similarly from control (normal) subjects, the individual has a high risk, or higher than normal risk, of developing UFC, whereas maintaining a normal range value of the "combined risk index" would indicate a low or minimal risk of developing U FC. In this embodiment, the threshold value would be or could be set by the combined risk index from one or more normal subjects.

[0051] In another embodiment, the value of the risk profile can be the collection of data from the individual measurements and need not be converted to a scoring system, such that the "risk profile value" is a collection of the individual measurements of the individual components of the profile.

[0052] In specific embodiments, a subject is diagnosed of having an increased risk of suffering from UFC if the subject's seven, six, five, four, three, two or even one of the components or factors herein are at abnormal levels.

[0053] If it is determined that a subject has an increased risk of developing U FC, the attending health care provider may subsequently prescribe or institute a treatment program. In this manner, the present invention also provides for methods of treating individuals for UFC. The attending healthcare worker may begin treatment, based on the subject's risk profile, before there are perceivable, noticeable or measurable signs of UFC in the individual.

[0054] Accordingly, the invention provides methods of treating UFC in a subject in need thereof. The treatment methods include obtaining a subject's risk profile as defined herein and prescribing a treatment regimen to the subject if the risk profile indicates that the subject is at risk of developing UFC.

[0055] The methods of treatment also include methods of monitoring the effectiveness of a treatment for U FC. Once a treatment regimen has been established, with or without the use of the methods of the present invention to assist in a diagnosis of a risk of developing U FC, the methods of monitoring a subject's risk profile over time can be used to assess the effectiveness of treatments for UFC. Specifically, the subject's risk profile can be assessed over time, including before, during and after treatments for U FC. The risk profile can be monitored, with, for example, the normalization or decline in the values of the profile over time being indicative that the treatment may be showing efficacy of treatment.

[0056] The present invention also provides kits that can be used in the methods of the present invention. Specifically, the present invention provides kits for assessing the increased risk of developing U FC, with the kits comprising one or more sets of antibodies that are immobilized onto a solid substrate and specifically bind to at least one of the factors or components listed herein. In specific embodiments, the kits comprise at least two, three, four, five, six or seven sets of antibodies immobilized onto a solid substrate, with each set corresponding to a factor.

[0057] The antibodies that are immobilized onto the substrate may or may not be labeled. For example, the antibodies may be labeled, e.g., bound to a labeled protein, in such a manner that binding of the specific protein may displace the label and the presence of the marker in the sample is marked by the absence of a signal. In addition, the antibodies that are immobilized onto the substrate may be directly or indirectly immobilized onto the surface. Methods for immobilizing proteins, including antibodies, are well-known in the art, and such methods may be used to immobilize a target protein, e.g., I L-10, or another antibody onto the surface of the substrate to which the antibody directed to the specific factor can then be specifically bound. In this manner, the antibody directed to the specific biomarker is immobilized onto the surface of the substrate for the purposes of the present invention.

[0058] The kits of the present invention may or may not include containers for collecting samples from the subject and one or more reagents, e.g., purified target biomarker for preparing a calibration curve. The kits may or may not include additional reagents such as wash buffers, labeling reagents and reagents that are used to detect the presence (or absence) of the label.

[0059] All patents and publications cited herein are incorporated by reference to the same extent as if each individual publication was specifically and individually indicated as having been incorporated by reference in its entirety. Examples

[0060] All patients undergoing exploratory laparotomy for blunt or penetrating trauma between September 2014 and June 2016 at a Level 1 trauma center were included. Serum and peritoneal fluid was collected at the initial laparotomy and all subsequent abdominal operations. Luminex and ProCT assays were performed on all specimens. A multiclass model was created to predict the outcomes of 3 groups of patients: 1) those managed with damage control laparotomy without achieving fascial closure, 2) those that achieved successful fascial closure and 3) those who dehisced after fascial closure. Constraint-based and local discovery learning algorithms from the "bnlearn" R package were used. The full dataset was then searched with a Bayesian network algorithm for a reduced variable set to build into the multiclass model. All models were assessed by receiver operator characteristic (ROC) curves and areas under curve (AUC).

[0061] Seventy-five patients were enrolled during the study period (67.7% penetrating, 33.3% blunt injury). Of these, 17.3% were managed with damage control laparotomy without achieving fascial closure, 73.3% underwent successful fascial closure, and 9.3% dehisced after fascial closure. There was no significant difference in age, gender, injury severity score, or initial base deficit among groups. The Bayesian network search algorithm selected Peritoneal Lavage ProCT, Serum ProCT, Peritoneal Lavage Interleukin (I L) 17 and Serum I L4. These variables were used to train a random forest classification model that performed well ( Kappa 0.41). The model accuracy rate of 0.64 outperformed the no information rate of 0.48. The sensitivity, specificity, and AUC for the 3 groups are summarized in Figure 1. Predicting successful fascial closure had the highest sensitivity (73%), while predicting failure of fascial closure had the highest specificity (92%). Comparing the AUC between groups revealed that predicting successful fascial closure performs the best compared to the other groups.

Claims

Claims:

1. A method of treating unsuccessful fascial closure after laparotomy in a human subject comprising:

(a) obtaining a first blood, serum or plasma sample from the human subject,

(b) determining levels of one or more polypeptides selected from the group consisting of procalcitonin (ProCT) and lnterleukin-4 ( IL-4) in the first blood, serum or plasma sample, and

(c) surgically closing the laparotomy within 24 hours in subjects with polypeptide levels about the same as human subjects with successful facial closure after laparotomy.

2. The method of claim 1 further comprising:

(d) obtaining a second blood, serum or plasma sample from the human subject at least 24 hours after obtaining the first blood, serum or plasma sample, and

(e) determining levels of one or more polypeptides selected from ProCT and IL-4 in the second blood, serum or plasma sample.

3. A method of treating unsuccessful fascial closure after laparotomy in a human subject comprising:

(a) obtaining a first peritoneal lavage sample from the human subject,

(b) determining levels of one or more polypeptides selected from ProCT and lnterleukin-17 (I L-17) in the first peritoneal lavage sample, and

(c) surgically closing the laparotomy within 24 hours in subjects with polypeptide levels about the same as human subjects with successful fascial closure after laparotomy.

4. The method of claim 3 further comprising:

(d) obtaining a second peritoneal lavage sample from the human subject at least 24 hours after obtaining the first peritoneal lavage sample, and

(e) determining levels of one or more polypeptides selected from ProCT and IL-17 in the second peritoneal lavage sample.

5. The method of claim 1 further comprising obtaining a first peritoneal lavage sample from the human subject, and determining levels of one or more polypeptides selected from ProCT and I L-17 in the first peritoneal lavage sample.

6. The method of claim 5 further comprising obtaining a second blood, serum or plasma

sample from the human subject, obtaining a second peritoneal lavage sample from the human subject, determining levels of one or more polypeptides selected from ProCT and I L-4 in the second blood, serum or plasma sample, determining levels of one or more polypeptides selected from ProCT and I L-17 in the second peritoneal lavage sample.

7. The method of any previous claim further comprising waiting at least two days before surgically closing the laparotomy.

8. The method of claims 2, 4, or 6 wherein the second sample is obtained 48 to 72 hours after obtaining the first sample.

9. The method of claims 2, 4, or 6 wherein the laparotomy is surgically closed after determining polypeptide levels in the second sample.

10. The method of any previous claim wherein the human subject has a trauma wound.

11. The method of claim 10 wherein the trauma wound is an abdominal wound.

12. A method of determining whether to surgically close a laparotomy in a human subject comprising:

(a) obtaining a blood, serum or plasma sample from the human subject; and

(b) measuring levels of one or more polypeptides selected from ProCT and IL-4 in the blood, serum or plasma sample;

wherein polypeptide levels about the same as human subjects with successful fascial closure after laparotomy indicate that the wound should be closed within 24 hours.

13. A method of determining whether to surgically close a laparotomy in a human subject comprising:

(a) obtaining a peritoneal lavage sample from the human subject; and

(b) measuring the levels of ProCT and IL-17 in the peritoneal lavage sample;

wherein polypeptide levels about the same as human subjects with successful fascial closure after laparotomy indicate that the laparotomy should be closed within 24 hours.

14. The method of claim 12 further comprising:

(c) obtaining a peritoneal lavage sample from the human subject; and

(d) measuring the levels of ProCT and IL-17 in the peritoneal lavage sample.

15. The method of any of claims 12-14 wherein the human subject has a trauma wound.

16. The method of any of claims 12-15 wherein the trauma wound is an abdominal wound.