The present invention relates to a method for predicting the outcome of a critically ill patient, said method comprising measuring the concentration of Chromogranin A or a fragment thereof in a biological sample obtained from said patient.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention relates to a method for predicting the outcome of a critically ill patient, said method comprising measuring the concentration of Chromogranin A or a fragment thereof in a biological sample obtained from said patient.

The present invention relates to a method for assessing the severity and/or morbidity of organ dysfunction, in particular MOF, in a patient, comprising measuring the concentration of chromogranin A (CGA) or a fragment thereof in a biological sample obtained from said patient.

The invention relates to the use of CGA or a fragment thereof as a marker of the severity and/or morbidity of critically illness.

The invention also relates to the use of CGA or a fragment thereof as a marker of the severity and/or morbidity of organ dysfunction, in particular MOF, in a patient.

DETAILED DESCRIPTION OF THE INVENTION

Diagnostic Methods and Kits

The present invention relates to a method for predicting the outcome of a critically ill patient, said method comprising measuring the concentration of Chromogranin A or a fragment thereof in a biological sample obtained from said patient.

The present invention relates to a method for assessing the severity and/or morbidity of organ dysfunction, in particular MOF, in a patient, comprising measuring the concentration of CGA or a fragment thereof in a biological sample obtained from said patient.

The invention relates to the use of CGA or a fragment thereof as a marker of the severity and/or morbidity of critically illness.

A further object of the invention relates to the use of CGA or a fragment thereof as a marker of the severity or morbidity of organ dysfunction, in particular MOF, in a patient.

Typically a patient according to the invention may be affected with sepsis or SIRS from non septic origin.

The inventors have found that (i) patients suffering from critical illnesses had a significant increase in blood CGA concentrations; (ii) a positive correlation between concentrations of CGA and standard biomarkers of inflammation existed in vivo; (iii) CGA concentrations were a further indication of predicting the risk of death for critically patients.

The inventors have shown a significant increase of blood CGA in acutely stressed patients compared with controls without SIRS (ie: healthy controls and self-poisoned patients without SIRS). In addition, there was a positive correlation between levels of CGA and inflammation markers. Finally, multivariable Cox proportional hazards regression demonstrated that level of CGA is a strong indicator of outcome in critically ill patients, in particular in patients affected with sepsis or systemic inflammatory response syndrome (SIRS) from non septic origin. The inventors have demonstrated that CGA significantly increases in non survivor groups that had more severe LODS or SAPS II scores. In addition, CGA concentrations were related to survival time.

The method of the invention may be thus useful for classifying a critically ill patient, in particular a patient affected with sepsis or systemic inflammatory response syndrome (SIRS) from non septic origin and then may be used to choose the accurate treatment in intensive care unit. For example, patients with a high score of severity may receive a more intensive treatment and attention compared to patient with a weak score. Such method may thus help the physician to make a choice on a therapeutic treatment which can accordingly consist in administering accurate drugs to the patients. Costs of the treatments may therefore be adapted to the severity and morbidity of the patients admitted in intensive care units, and accordingly the method of the invention may represent a useful tool for the management of such units. Finally, the method of the invention may be applied for monitoring the therapeutic outcome of a critically ill patient, in particular of a patient affected with sepsis or systemic inflammatory response syndrome (SIRS) from non septic origin.

The inventors have surprisingly demonstrated that Chromogranin A (CGA) is a marker for severity or morbidity of organ dysfunction, in particular MOF, in patients affected with sepsis or systemic inflammatory response syndrome (SIRS) from non septic origin. The ROC analysis has further demonstrated that blood CGA concentration on admission is a reliable predictor of mortality for sepsis or systemic inflammatory response syndrome (SIRS) from non septic origin, as efficient equivalent as the SAPS score. Therefore, in contrast to SAPS which requires the analysis of 17 parameters, CGA and fragments thereof represent a unique and accurate tool of determining the severity of organ dysfunction in patient affected with sepsis or systemic inflammatory response syndrome (SIRS) from non septic origin. Moreover, CGA may be easily and swiftly quantified.

Typically, the method of the invention may comprise a step of comparing the concentration of CGA or a fragment thereof with a predetermined threshold value. Said comparison is indicative of the outcome of a critically ill patient and of the severity or morbidity of organ dysfunction or MOF in said patient.

Typically said predetermined threshold value may be determined by taking into account the patient's known diseases and/or treatments which are known to elevate the blood concentration of CGA.

Examples of diseases and/or treatments known to elevate the blood concentration of CGA are pheochromocytomas, carcinoid tumors, neuroblastomas, neuroendocrine tumors, neurodegenerative diseases, chronic heart failure, acute myocardial infarction, complicated myocardial infarction, chronic renal failure, hepatic failure, ongoing steroids treatment, or ongoing proton pump inhibitors treatment, or surgical intervention.

In an embodiment of the invention, said patient is not known for being affected with a disease known to elevate the blood concentration of CGA, such as pheochromocytomas, carcinoid tumors, neuroblastomas, neuroendocrine tumors, neurodegenerative diseases, chronic heart failure, acute myocardial infarction, complicated myocardial infarction, chronic renal failure or hepatic failure.

In an embodiment of the invention, a patient according to the invention is not known for undergoing or having recently undergone a treatment known to elevate the blood concentration of CGA, such as steroids treatment, proton pump inhibitors treatment or a surgical intervention.

In an embodiment of the invention, a patient according to the invention is not a patient requiring a surgical intervention.

Once the biological sample from the patient is prepared, the concentration of CGA or a fragment thereof may be measured by any known method in the art.

For example, the concentration of CGA or a fragment thereof may be measured by using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, high performance liquid chromatography (HPLC), size exclusion chromatography, solid-phase affinity, etc.

In a particular embodiment, such methods comprise contacting the biological sample with a binding partner capable of selectively interacting with CGA or a fragment thereof present in the biological sample.

The binding partner may be generally an antibody that may be polyclonal or monoclonal, preferably monoclonal. Polyclonal antibodies directed against CGA or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against CGA can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler et al. Nature. 1975; 256(5517):495-7; the human B-cell hybridoma technique (Cote et al Proc Natl Acad Sci USA. 1983; 80(7):2026-30); and the EBV-hybridoma technique (Cole et al., 1985, In Monoclonal Antibodies and Cancer Therapy (Alan Liss, Inc.) pp. 77-96). Alternatively, techniques described for the production of single chain antibodies (see e.g. U.S. Pat. No. 4,946,778) can be adapted to produce anti-CGA, single chain antibodies. Antibodies useful in practicing the present invention also include anti-CGA fragments including but not limited to F(ab').sub.2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab').sub.2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to CGA. For example, phage display of antibodies may be used. In such a method, single-chain Fv (scFv) or Fab fragments are expressed on the surface of a suitable bacteriophage, e.g., M13. Briefly, spleen cells of a suitable host, e.g., mouse, that has been immunized with a protein are removed. The coding regions of the VL and VH chains are obtained from those cells that are producing the desired antibody against the protein. These coding regions are then fused to a terminus of a phage sequence. Once the phage is inserted into a suitable carrier, e.g., bacteria, the phage displays the antibody fragment. Phage display of antibodies may also be provided by combinatorial methods known to those skilled in the art. Antibody fragments displayed by a phage may then be used as part of an immunoassay.

In another embodiment, the binding partner may be an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by EXponential enrichment (SELEX) of a random sequence library, as described in Tuerk et al. (1990) Science, 249, 505-510. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena (1999) Olin Chem. 45(9):1628-50. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by a platform protein, such as E. coli Thioredoxin A, that are selected from combinatorial libraries by two hybrid methods (Colas et al. (1996). Nature, 380, 548-50).

The binding partners of the invention such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.

As used herein, the term "labeled", with regard to the antibody, is intended to encompass direct labeling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be labeled with a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as I123, I124, In111, Re186, Re188.

The aforementioned assays generally involve the bounding of the binding partner (ie. Antibody or aptamer) in a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against CGA or a fragment thereof. A biological sample containing or suspected of containing CGA or a fragment thereof is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

Different immunoassays, such as radioimmunoassay or ELISA have been described in the art. For example, document U.S. Pat. No. 4,758,522 describes an immunoassay of CGA in which CGA is measured by competitive assay with radiolabelled CGA for the sites of an anti-human CGA antibody. Syversen et al (Acta Oncol. 1993; 32(2):161-5) have described an assay of chromogranin A with the ELISA technique using an antibody directed against a C-terminal fragment of CGA. Corti et al. (Br J. Cancer. 1996 April; 73(8):924-32) have also described two assays of CGA using monoclonal antibodies directed human CGA. Document U.S. Pat. No. 6,632,624 further describes an immunoassay in which at least one monoclonal antibody specifically binds to an epitope corresponding to amino acids 145 to 234 of human CGA. Tests for detecting CGA in neuroendocrine tumors have been described by Huttner et al. (1991) Trends Biochem. Sci. 16, 27-30, Winkler et al. (1992) Neuroscience 49, 497-528, Deftos (1991) Endocr Rev 12, 181-187 and Degorce et al., (1999) Br. J. Cancer 79, 65-71. Radioimmunoassays are commercially available from CIS BIO International (30200 Bagnols/Ceze--France), Alpco Diagnostics (Windham, N.H. 03087, USA), and Immuno-Biological Laboratories (Minneapolis, Minn. 55432, USA). ELISA kits for CGA are also commercially available from Alpco Diagnostics (Windham, N.H. 03087, USA) and Cosmo Bio, LTD (Tokyo 135-0016, Japan).

Another interesting approach concerns Bioplex (Biorad) using several specific CGA antibodies.

Measuring the concentration of CGA (with or without immunoassay-based methods) may also include separation of the proteins: centrifugation based on the protein's molecular weight; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid-phase affinity based on the protein's affinity for the particular solid-phase that is use. Once separated, CGA may be identified based on the known "separation profile" e.g., retention time, for that protein and measured using standard techniques. Alternatively, the separated proteins may be detected and measured by, for example, a mass spectrometer.

Yet another object of the invention relates to a kit, comprising means for measuring the concentration of CGA. The kit may include an antibody, or a set of antibodies as above described. In a particular embodiment, the antibody or set of antibodies are labelled as above described. The kit may also contain other suitably packaged reagents and materials needed for the particular detection protocol, including solid-phase matrices, if applicable, and standards. The kit may also contain one or more means for the detection of marker of organ dysfunction or infection. Typically the kit may also contain means for the detection of C reactive protein (CRP) and/or means for the detection procalcitonin (PCT) and/or means for the detection white blood cells.

A further object of the invention relates to the use of CGA or a fragment thereof as a marker of infection in a patient. An embodiment of the invention relates to a method for diagnosing an infection in a patient, said method comprising measuring the concentration of Chromogranin A or a fragment thereof in a biological sample obtained from said patient. Typically the method may further comprise detecting one or more other markers of infection such as C reactive protein (CRP) and/or procalcitonin (PCT) and/or white blood cells in a biological sample obtained from said patient.
 

Claim 1 of 4 Claims

1. A method for predicting the survival outcome of a critically ill patient, said method comprising measuring the concentration of Chromogranin A (CGA) in a biological sample obtained from said patient wherein an elevated level of CGA in said critically ill patient as compared to the level of CGA in control patient predicts a shorter survival for said critically ill patient as compared to said control patient wherein said patient is affected with sepsis or systemic inflammatory response syndrome (SIRS) from non septic origin.
 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.