METHODS FOR DIAGNOSIS OF PROLIFERATIVE DISEASES

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a method and a diagnostic system for diagnosing glioma.BACKGROUND OF THE INVENTION

[0002] Proliferative disorders, such as cancer, are caused by uncontrolled and unregulated cellular proliferation. Such cellular proliferation can lead to the formation of tumours in the relevant subjects.

[0003] Typically, tumours, such as brain tumours, are initially clinically identified within a subject by way of various well known pre-screening imaging techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), X-Rays, and positron emission tomography (PET). Such imaging techniques are, however, expensive to deploy given the high cost of both the equipment itself and the human resources required to operate it. Some such imaging techniques require complex operation by highly qualified professionals, and some require time consuming analysis before conclusions can be drawn. Moreover, such techniques seldom, if ever, distinguish between benign and malignant tumours. As such, a final biopsy is always required to confirm the malignancy or benignity of a given tumour.

[0004] Biopsies require invasive surgery to extract a relevant tissue sample. In the case of brain tumours, biopsies generally require drilling into the subject's skull, which is a highly dangerous and skilled surgical operation. The subject having undergone such a biopsy is then typically hospitalised for two to three days, which presents an undesirable care burden. Once the biopsy has been successfully performed, it can take a significant period of time before the malignancy or benignity of the relevant tumour is actually determined.

[0005] It is therefore highly desirable to provide a pre-screening tool that is cost-effective, requires minimal human resource and skill to operate, and does not involve time consuming analysis. It is moreover desirable to provide a pre-screening technique that facilitates relatively fast determination of malignancy or benignity of tumours with a reasonably high degree of accuracy, and without the drawbacks inherent with biopsies.

[0006] In recent times, various biomarkers within the blood have been identified as useful indicators of particular diseases. For instance, cytokines, chemokines, and growth factors are cell signalling proteins that mediate a range of physiological responses, and are associated with various diseases. Such molecules are generally detected by either bioassay or immunoassay, both of which can be time consuming given that often only one analyte may be analysed at a time. However, in more recent times, magnetic bead-based multiplex assays designed to measure multiple cytokines, chemokines, and growth factors in diverse matrices like serum, plasma, and tissue culture supernatants, have become more readily available with kits such as Bio-Plex Pro ™< (see Bio-Plex Pro ™< Assay Handbook - http: / / www.bio-rad.com / webroot / web / pdf / lsr / literature / 10014905.pdf). However, the complexities associated with the correlation of particular biomarkers with particular diseases has retarded developments in the medical diagnostics field, and such correlations are inherently unpredictable at present. Moreover, such assaying still requires a reasonable level of skill, and such assays also destroy the sample in question such that repeat assays on the same sample are not possible. Validation of results is thus more difficult.

[0007] AYSEGUL ILHAN-MUTLU ET AL: "Exploratory investigation of eight circulating plasma markers in brain tumor patients", NEUROSURGICAL REVIEW, SPRINGER, BERLIN, DE, vol. 36, no. 1, 5 July 2012 (2012-07-05), pages 45 - 56, XP035159037, ISSN: 1437-2320, DOI: 10.1007 / S10143-012-0401-6, relates to investigations of eight circulating plasma markers in brain tumor patients.

[0008] XU BAOGANG J ET AL: "Identification of blood protein biomarkers that aid in the clinical assessment of patients with malignant glioma", INTERNATIONAL JOURNAL OF ONCOLOGY, SPANDIDOS: ATHENS, GR, vol. 40, no. 6, 1 June 2012 (2012-06-01), pages 1995 - 2003, XP009162919, ISSN: 1791-2423, DOI: 10.3892 / IJO.2012.1355, relates to identification of blood protein biomarkers in the clinical assessment of malignant glioma.

[0009] US20110027797A1 (Kumaravel Somasundaram) relates to methods of diagnosing higher- and lower-grade Astrocytoma using biomarkers.

[0010] K RAE ET AL: "Follistatin serum concentrations during full-term labour in women significant differences between spontaneous and induced labour", REPRODUCTION, vol. 134, no. 5, 26 October 2007 (2007-10-26), GB, pages 705 - 711, XP055306869, ISSN: 1470-1626, DOI: 10.1530 / REP-07-0208, relates to follistatin serum concentrations during full-term labour in women, and the variations thereof between spontaneous and induced labour.

[0011] P. SREEKANTHREDDY ET AL: "Identification of Potential Serum Biomarkers of Glioblastoma: Serum Osteopontin Levels Correlate with Poor Prognosis", CANCER EPIDEMIOLOGY, BIOMARKERS AND PREVENTION., vol. 19, no. 6, 1 June 2010 (2010-06-01), US, pages 1409 - 1422, XP055306880, ISSN: 1055-9965, DOI: 10.1158 / 1055-9965.EPI-09-1077, relates to potential serum biomarkers of glioblastoma.

[0012] D F ZHANG ET AL: "Expression of activin A and follistatin in glioblastoma and their effects on U87 in vitro", THE JOURNAL OF INTERNATIONAL MEDICAL RESEARCH, 1 July 2010 (2010-07-01), England, pages 1343 - 1353, XP055306883, Retrieved from the Internet <URL:http: / / imr.sagepub.com / content / 38 / 4 / 1343.full.pdf> [retrieved on 20160928], relates to the expression of activin A and follistatin in glioblastoma and their effects on U87 in vitro.

[0013] US20120064529A1 (Predictive Biosciences, Inc.) relates to methods for detecting a target nucleic acid and a target protein in a single assay.

[0014] POUNEH KERMANI ET AL: "BDNF: A Newly Described Mediator of Angiogenesis", 1 May 2007 (2007-05-01), XP055392322, Retrieved from the Internet <URL:https: / / www.ncbi.nim.nih.gov / pmc / articles / PMC2268985 / pdf / nihms-23333.pdf> [retrieved on 20170719], relates to a mediator of angiogenesis.

[0015] XIN ZHOU ET AL: "Conceptual and methodological issues relevant to cytokine and inflammatory marker measurements in clinical research", PUBLISHED IN FINAL EDITED FORM AS: CURR OPIN CLIN NUTR METAB CARE, 1 September 2010 (2010-09-01), pages 541 - 547, XP055756364, Retrieved from the Internet <URL:https: / / www.ncbi.nim.nih.gov / pmc / articles / PMC2955626 / pdf / nihms243735.pdf> [retrieved on 20201203], DOI: 10.1097 / MCO, relates to conceptual and methodological issues relevant to cytokine and inflammatory marker measurements in clinical research.

[0016] LENG S X ET AL: "ELISA and multiplex technologies for cytokine measurement in inflammation and aging research", vol. 63, no. 8, 1 August 2008 (2008-08-01), pages 879 - 884, XP008154409, ISSN: 1079-5006, Retrieved from the Internet <URL:http: / / biomedgerontology.oxfordjournals.org / content / 63 / 8 / 879.abstract> DOI: 10.1093 / GERONA / 63.8.879, relates to ELISA and multiplex technologies for cytokine measurement in inflammation and aging research.

[0017] LUNDBLAD R: "ISPUB Considerations for the Use of Blood Plasma and Serum for Proteomic Analysis", THE INTERNET JOURNAL OF GENOMICS AND PROTEOMICS, 1 January 2003 (2003-01-01), XP055756353, Retrieved from the Internet URL:https: / / print.ispub.com / api / 0 / ispub-article / 3649, relates to ISPUB considerations for the use of blood plasma and serum for proteomic analysis.

[0018] It is therefore an object of the present invention to solve at least one of the problems inherent with the prior art. Another object is to provide a simple, reliable, and cost-effective point-of-care diagnostic method that requires minimal human resource and skill to operate, is non-time consuming, and which facilitates rapid determination of malignancy / benignity of tumours with a reasonably high degree of accuracy.SUMMARY OF THE INVENTION

[0019] According to an aspect, the present invention provides a method of diagnosing glioma in a subject as set forth in appended claim 1. This method comprises assaying a blood plasma sample of the subject in respect of two or more biomarkers selected from the group consisting of Angiopoietin, Follistatin, HGF, IL-8, Leptin, PDGF-BB, PECAM-1, PDGF-AA, sHER2 neu, sIL-6R alpha, prolactin, sVEGFR1, G-CSF, and FGF, wherein one of the two or more biomarkers is prolactin; and correlating elevated or reduced levels of each of the two or more biomarkers, whether relative to a predetermined threshold or relative to each other, with a determination of the presence of glioma in the subject.

[0020] According to an aspect, the present invention provides a diagnostic system for diagnosing glioma in a subject as set forth in appended claim 11. This diagnostic system comprises: a device configured to receive a blood plasma sample from the subject and configured to assay the blood plasma sample in respect of the two or more biomarkers defined in respect of the aforementioned method of diagnosing glioma; and a computer configured to correlate or facilitate correlation of the amounts of the two or more biomarkers within the blood plasma sample with a determination of the presence of glioma in the subject, wherein the computer is configured to carry out the step of correlating elevated or reduced levels of each of the two or more biomarkers, whether relative to a predetermined threshold or relative to each other, with a determination of the presence of glioma in the subject, as defined in the aforementioned method of diagnosing glioma.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figures 1 to 7 show graphical representations of the "control mean" (light grey) and "glioma mean" (dark grey), and also error bars, in relation to IL-8, Angiopoietin, Follistatin, HGF, Leptin, PDGF-BB, and PECAM-1 respectively. Figures 7A to 7F show graphical representations of the "control mean" (dark grey - left), "low grade glioma mean" (light grey - middle), and "high grade glioma mean" (medium grey - right) and also error bars, in relation to FGF, G-CSF, sHER2neu, sIL-6Ralpha, Prolactin, and sVEGFR1 respectively. Figure 8 is a scatter-graphical correlation chart for PECAM-1 and PDGF-BB showing the relationship between PECAM-1 and PDGF-BB levels in the 50 glioma patients, and demonstrating a degree of linearity and a correlation coefficient of 0.45. Figures 8A-8G shows photographic immunohistochemical comparisons between glioma and non-cancerous brain tissues, namely: a) glioma tumour section x40 magnification showing positively staining and non-staining tumour cells; b) glioma tumour section x40 magnification showing negatively staining blood vessels; c) non-cancerous brain tissue x40 magnification showing negatively staining blood vessel; d) glioma tumour section x40 magnification showing interstitial staining; e) glioma tumour section x40 magnification showing interstitial staining, particularly of axonal tracts; f) non-cancerous brain tissue x40 magnification showing negatively staining blood vessel; g) choroid plexus tissue showing positive cytoplasmic staining. DETAILED DESCRIPTION OF THE INVENTION Definitions

[0022] Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.

[0023] Herein, "diagnosis" generally includes a determination of the presence of glioma.

[0024] Herein, "plasma" refers to the straw-colored / pale-yellow liquid component of blood that normally holds the blood cells in whole blood in suspension. It makes up about 55% of total blood volume. It is the intravascular fluid part of extracellular fluid (all body fluid outside of cells). It is mostly water (93% by volume) and contains dissolved proteins (major proteins are fibrinogens, globulins and albumins), glucose, clotting factors, mineral ions (Na +< , Ca ++< , Mg ++< , HCO 3 -< Cl -< etc.), hormones and carbon dioxide (plasma being the main medium for excretory product transportation). It is to be noted that, for plasma samples, both EDTA plasma and citrate plasma are suitable, whereas heparin plasma is less preferred, since this can absorb certain cytokines.

[0025] "Cytokines" are well known in the art as cell-signaling protein molecules that are secreted by numerous cells and are a category of signaling molecules used extensively in intercellular communication. Cytokines can be classified as proteins, peptides, or glycoproteins; the term "cytokine" encompasses a large and diverse family of regulators produced throughout the body by cells of diverse embryological origin. Some "cytokines" may also be considered "angiogenesis factors", and visa versa.

[0026] "Angiogenesis Factors" are well known in the art as angiogenic growth factors. In the context of the present invention, "cytokines" are generally considered collectively with "angiogenesis factors" given their combined service as biomarkers for glioma, as demonstrated in the Examples and throughout the specification.

[0027] Herein, references to an "assay" or "assaying" includes any form of analysis, including standard biological assays (e.g. bioassays, immunoassays, etc.).

[0028] As used herein, a "subject" refers to an animal, preferably a mammal. In preferred embodiments, the subject is a human subject. In other embodiments, the subject is a non-human mammal, including but are not limited to, dog, cat, horse, etc.Proliferative Disorder - Glioma

[0029] The methods and diagnostic systems of the present invention pertain to glioma.

[0030] The three main types of malignant glioma are astrocytomas, ependymomas and oligodendrogliomas. The diagnostic methods of the invention may apply to all these types of glioma. A tumour with a mixture of the histological features present in the main three is known as a mixed glioma, which the present invention may also serve to diagnose. The table below shows the sub-types of high grade and low-grade gliomas. General Tumour Grade WHO Grade Grade Sub-type Low Grade IPilocytic astrocytomaIIOligodendrogliomaIIAstrocytomaHigh Grade IIIAnaplastic astrocytomasIIIOligodendrogliomasIVGlioblastoma multiforme

[0031] In a particular embodiment, the glioma is either a low grade or high grade glioma. In a particular embodiment, the glioma is any one of Pilocytic astrocytoma, Oligodendroglioma, Astrocytoma, Anaplastic astrocytomas, Oligodendrogliomas, Glioblastoma multiforme glioma sub-types.

[0032] In a particular embodiment, the glioma is a Grade III or Grade IV glioma.Subjects (Patients)

[0033] The subject is suitably an animal, preferably a mammal. In preferred embodiments, the subject is a human subject. In other embodiments, the subject is a non-human mammal, including but are not limited to, dog, cat, horse, etc.

[0034] The subject suitably has or is suspected as having glioma.

[0035] The subject is suitably a glioblastoma or a gliosarcoma patient. In a particular embodiment, the subject is a glioblastoma patient.Blood Plasma Sample

[0036] In the context of the methods and diagnostic systems of the present invention, a blood plasma sample is used.Cytokines and Angiogenesis Factors

[0037] In the context of the methods and diagnostic systems of the present invention, the two or more biomarkers are selected from the group consisting of Angiopoietin, Follistatin, HGF, IL-8, Leptin, PDGF-BB, PECAM-1, PDGF-AA, sHER2 neu, sIL-6R alpha, prolactin, sVEGFR1, G-CSF, and FGF, wherein one of the two or more biomarkers is prolactin. Such biomarkers are cytokines and / or angiogenesis factors.

[0038] All the abovementioned abbreviations are outlined below. In any event, all of the abovementioned cytokines and angiogenesis factors are well known in the art without further elaboration, and are available commercially or in assay kits.Analysis of Blood Plasma Sample - Diagnosis

[0039] The methods of diagnosing glioma in a subject, as described herein, all involve analysis of a blood plasma sample in respect of the aforesaid two or more biomarkers.Assaying a Blood Plasma Sample

[0040] Assaying the blood plasma sample suitably involves determining the levels of the two or more biomarkers within the blood plasma sample.

[0041] The levels (or calibrated / normalised levels) of the two or more biomarkers may be assessed, for instance, against a predetermined threshold (e.g. determined by prior studies of cytokine / angiogenesis factor levels in blood plasma samples of a representative cross-section of subjects with and without glioma) for each of the two or more biomarkers or relative to each other (e.g. comparing the relative levels / profile of the cytokines / angiogenesis factors concerned). Such an assessment may then be correlated with a diagnosis. In particular, observation of elevated or reduced levels of each of the two or more biomarkers, whether relative to a predetermined threshold or relative to each other, may be correlated with a glioma diagnosis.

[0042] In a particular embodiment, the blood plasma sample(s) are assayed with an immunoassay, for instance based on an antigen-antibody response.

[0043] Assaying the blood plasma sample may involve any suitable assay known in the art. Each of the two or more biomarkers may be assayed for individually, optionally in series. As such, a blood plasma sample may be split into multiple aliquots for testing. Alternatively, each of the two or more biomarkers may be assayed in parallel (e.g. as multiple aliquots). Alternatively, each of the two or more biomarkers may be assayed in parallel in the sample assay (i.e. with a single blood plasma sample), for instance via a multiplex assay.

[0044] In a particular, the blood plasma sample(s) are assayed using a magnetic bead-based multiplex assay designed to measure multiple cytokines and / or angiogenesis factors. The multiplexing feature makes it possible to quantitate the level of multiple proteins in a single well, in just 3 hrs, using as little as 12.5 µl of plasma. Suitable assay kits include the Bio-Plex ™< and Bio-Plex ™< Pro systems, which incorporate magnetic beads into their design. The magnetic beads allow for the option of using magnetic separation during wash steps instead of vacuum filtration. Magnetic separation allows for greater automation without significant alterations to the standard Bio-Plex assay protocol.

[0045] The assay suitably employs a plurality of fluorescently dyed beads (e.g. xMAP technology) to simultaneously detect multiple cytokines and / or angiogenesis factors in a single assay (e.g. a single well). As such, two or more biomarkers may be the subject of analysis. In a particular embodiment, the up to 100 unique fluorescently dyed beads are used for cytokine / angiogenesis factor detection.

[0046] The assay suitably employs a flow cytometer with two lasers and associated optics to measure the different cytokines / angiogenesis factors bound to the surface of the beads.

[0047] The assay suitably employs a diagnostic kit with a (high-speed) digital signal processor that efficiently manages the fluorescent data.

[0048] The bead-based assays suitably operate in a manner similar to a capture sandwich immunoassay. For instance, an antibody directed against the desired cytokine and / or angiogenesis factor targets is suitably covalently bound to internally dyed beads. During the assay, the beads are suitably contacted with the relevant blood plasma sample to facilitate reaction between the covalently bound antibody and the target cytokines and / or angiogenesis factors. After a sufficient contact time, the beads are suitably washed (optionally several times) to remove unbound protein. Thereafter, a biotinylated detection antibody specific to an epitope different from that of the capture antibody is suitably added to the bead reaction mixture. This suitably produces a sandwich of antibodies around the cytokine / angiogenesis factor targets. A reporter complex (e.g. streptavidin-phycoerythrin (streptavidin-PE)) is then suitably added to bind to the biotinylated detection antibodies on the bead surface.

[0049] Data is suitably acquired from the bead reaction mixture using a suitable reader system. In a particular embodiment, the data is acquired using the Bio-Plex system (or Luminex system), a dual-laser, flow-based microplate reader system. The bead reaction mixture is suitably drawn up into the reader system. Lasers and associated optics suitably detect the internal fluorescence of the individual dyed beads as well as the fluorescent reporter signal on the bead surface. This suitably identifies each assay and reports the level of cytokine / angiogenesis factor target in the sample. Intensity of fluorescence detected on the beads indicates the relative quantity of target cytokines and / or angiogenesis factor molecules in the tested samples. A digital processor suitably manages the data output, which is suitably further analyzed and presented as fluorescence intensity (FI) and target concentration data, potentially using Bio-Plex Manager ™< software.

[0050] The levels of the two or more biomarkers can then be used to determine a glioma diagnosis, as described above, whether manually or automatically (i.e. through the data being directly processed by a computer as defined herein).Diagnostic System

[0051] The present invention provides a diagnostic system for diagnosing glioma in a subject. This diagnostic system is defined hereinbefore and is as set forth in appended claim 11. This diagnostic system comprises: a device configured to receive a blood plasma sample from the subject and configured to assay the blood plasma sample in respect of two or more biomarkers defined in respect of the method of diagnosing glioma of the present invention; and a computer configured to correlate or facilitate correlation of the amounts of the two or more biomarkers within the blood plasma sample with determination of the presence of glioma in the subject, wherein the computer is configured to carry out the step of correlating elevated or reduced levels of each of the two or more biomarkers, whether relative to a predetermined threshold or relative to each other, with a determination of the presence of glioma in the subject, as defined in the method of diagnosing glioma.

[0052] Suitably the device for correlating or facilitating correlation of the results comprises the computer or is in communication with the computer (e.g. whether wired or wireless).EXAMPLE - Assay of a Blood Plasma Sample

[0053] In the present example, cytokine and angiogenesis factor assays were performed upon blood plasma samples using the magnetic bead-based multiplex assays provided by a Bio-Plex Pro ™< Assay kit. All the relevant protocols, which were duly followed in the present example, are set forth in the Instruction Manual entitled "Bio-Plex Pro ™< Assays Cytokine, Chemokine, and Growth Factors Instruction Manual" available from Bio-Rad Laboratories, Inc at the website www.bio-rad.com, and in particular at http: / / www.bio-rad.com / webroot / web / pdf / lsr / literature / 10014905.pdf. The protocols of this Instruction Manual were followed in relation to "Bio-Plex Pro ™< Human, Mouse, and Rat Cytokine Assays". The Bio-Plex ™< system was prepared as described in the Instruction Manual, suitably calibrated, and validated as described. The magnetic beads present in the 96-well Bio-Plex Pro flat-bottom plates were washed via magnetic separation using the magnetic setting of the Bio-Plex Pro wash station. The 96-well Bio-Plex Pro flat-bottom plates were laid out appropriately, with wells assigned appropriately. Appropriate standards, supplied with the Bio-plex system, were prepared in accordance with the Protocols set forth in the Instruction Manual.

[0054] As described in the Instruction Manual, The Bio-Plex ™< suspension array system is built around the three core elements of xMAP technology: Fluorescently dyed microspheres (also called beads), each with a distinct color code or spectral address to permit discrimination of individual tests within a multiplex suspension. This allows simultaneous detection of more than 100 different types of molecules in a single well of a 96-well microplate A dedicated flow cytometer with two lasers and associated optics to measure the different molecules bound to the surface of the beads A high-speed digital signal processor that efficiently manages the fluorescence data

[0055] Bio-Plex Pro ™< cytokine, chemokine, and growth factor assays are essentially immunoassays formatted on magnetic beads. The assay principle is similar to that of a sandwich ELISA (Figure 1). Capture antibodies directed against the desired biomarker are covalently coupled to the beads. Coupled beads react with the sample containing the biomarker of interest. After a series of washes to remove unbound protein, a biotinylated detection antibody is added to create a sandwich complex. The final detection complex is formed with the addition of streptavidin-phycoerythrin (SA-PE) conjugate. Phycoerythrin serves as a fluorescent indicator, or reporter.

[0056] As also explained in the Instruction Manual, data from the reactions are acquired using a Bio-Plex system or similar_Luminex-based reader. When a multiplex assay suspension is drawn into the Bio-Plex 200 reader for example, a red (635 nm) laser illuminates the_fluorescent dyes within each bead to provide bead classification and_thus assay identification. At the same time, a green (532 nm) laser excites PE to generate a reporter signal which is detected by a photomultiplier tube (PMT). A high-speed digital processor manages data output and Bio-Plex Manager ™< software presents data as Median Fluorescence Intensity (MFI) as well as concentration (pg / mL). The concentration of analyte bound to each bead is proportional to the median fluorescence intensity (MFI) of reporter signal.

[0057] The Instruction Manual summarises the initial preparation for the assays as follows: 1. Plan the plate layout 2. Start up / warm up the Bio-Plex system (up to 30 min) Meanwhile, equilibrate assay reagents to room temperature (RT) Begin to thaw samples 3. Prime wash station or calibrate vacuum manifold 4. Calibrate the system (now, or later during an incubation) 5. Reconstitute a single vial of standards in 500 µl of the appropriate diluent, vortex and incubate on ice (30 min) For serum and plasma samples (as per the present example), use Bio-Plex standard diluent 6. Prepare the 8 point standard dilution series and blank. Add 72 µl diluent to tube S1, and 150 µl diluent to tubes S2-8 and blank. Transfer 128 µl reconstituted standard into S1 Then serially dilute 4 fold from S1 thru S8 by transferring 50 µl between tubes. Vortex between transfers 7. Once thawed, prepare 1x samples Dilute serum, plasma and lysates in Bio-Plex sample diluent 8. Prepare 1x coupled beads in assay buffer, protect from light From 10x stock: Add 575 µl beads to 5,175 µl buffer From 20x stock: Add 288 µl beads to 5,472 µl buffer 9. Make sure samples and standards are at RT before dispensing

[0058] The Instruction Manual summarises the running of the assays as follows: 1. Prewet filter plate with 100 µl assay buffer (skip for flat bottom) 2. Add 50 µl of 1x beads to the assay plate 3. Wash 2 times with 100 µl wash buffer 4. Add 50 µl samples, standards, blank, controls 5. Cover and incubate in the dark at RT with shaking at 300 RPM 30 min - Human Group I,II and Mouse Group I,II With 10 min remaining, prepare 1x Detection Ab in detection antibody diluent. From 10x stock: Add 300 µl Ab to 2,700 µl diluent From 20x stock: Add 150 µl Ab to 2,850 µl diluent 6. Wash 3 times with 100 µl wash buffer 7. Add 25 µl of detection antibody 8. Cover and incubate in the dark at RT with shaking at 300 RPM 30 min - Human Group I,II; Mouse Group I,II Meanwhile, prepare software protocol; enter normalized standard S1 values With 10 min remaining, prepare 1x SA-PE in assay buffer, From 100x stock: Add 60 µl SA-PE to 5,940 µl assay buffer. Protect from light 9. Wash 3 times with 100 µl wash buffer 10. Add 50 µl of strepavidin-PE 11. Cover and incubate in the dark at RT with shaking at 300 RPM 10 min - Human Group I,II; Mouse Group I,II 12. Wash 3 times with 100 µl wash buffer 13. Resuspend beads in 125 µl assay buffer, shake at 1100 RPM for 30 sec 14. Read plate Low PMT (Low RP1) - Human group I,II; Mouse group I,II

[0059] In accordance with the Instruction Manual, the reagents supplied with the Bio-Plex Pro ™< assay kits for human, mouse, and rat cytokine assays include (Table 1):

[0060] In accordance with the Instruction Manual, the testable cytokines include (Table 2):

[0061] However, additional cytokines and angiogenesis factors were in fact tested, and the relevant standards and protocols developed accordingly. These additional cytokines and angiogenesis factors are detailed in the results section.Whole Blood Sampling

[0062] Whole blood samples were collected from 50 glioma patients and 27 healthy subjects.Preparation of Blood Plasma Samples from the Whole Blood Samples

[0063] Blood plasma samples for each of the 50 glioma patients and 27 healthy subjects were prepared by adding the corresponding fresh whole blood sample to a tube containing an anticoagulant, and spinning the tube at 13,200 rpm for 10 min at 4°C until the blood cells fell to the bottom of the tube to clear the samples of precipitate. The blood plasma was then poured or drawn off. The resulting blood plasma had a density of approximately 1025 kg / m 3< , or 1.025 kg / l. The blood plasma samples were then either assayed immediately or otherwise aliquoted and stored in single use aliquots at -70°C for later use, though repeated freeze / thaw cycles were avoided.

[0064] Before conducting the assays, 1 volume of plasma sample was diluted with 3 volumes of sample diluents (for example, 50 µL sample + 150 µL sample diluents).Preparation of Coupled Beads

[0065] The preparation of coupled beads is now described using the protocols espoused in the Bio-Plex ™< Pro instruction manual.

[0066] One tube of coupled beads is included with each kit. Instructions are provided for diluting the coupled beads to a 1x concentration.

[0067] When using 10-pack reagents, ensure that only the required volumes of coupled beads, detection antibodies, streptavidin-PE, and buffers have been removed from the tubes or bottles. For example, transfer a one-time volume of assay buffer, sufficient to perform all steps of the assay procedure (that is, prewetting the filter plate, diluting coupled beads, diluting streptavidin-PE, and resuspending the beads) into a 50 ml reservoir. 1. Use the Calculation Worksheet shown below to calculate the volume of coupled beads and assay buffer needed. 2. Add the required volume of assay buffer to a 15 ml polypropylene tube. 3. Vortex the coupled beads at mid speed for 30 sec. Carefully open the cap and pipet any liquid trapped in the cap back into the tube. This is important to ensure maximum bead recovery. Do not centrifuge the vial; doing so will cause the beads to pellet. 4. Pipet the required volume of stock coupled beads into the 15 ml tube containing assay buffer to dilute the coupled beads to a 1x concentration. Each well requires either 5 µl coupled beads (10x) or 2.5 µl coupled beads (20x) adjusted to a final volume of 50 µl using assay buffer. Refer to the example bead calculations in Tables 3-6 below, which include a 20% excess to compensate for transfer loss. 5. Protect the beads from light with aluminum foil. Equilibrate at room temperature for 20 min prior to use. Magnetic bead-based multiplex assay

[0068] The assays were then run as described in the Bio-Plex ™< Pro instruction manual (as also set forth below).

[0069] Bring all buffers, diluted standards, diluted coupled beads, and samples to room temperature prior to use. To ensure optimal performance, pipet carefully (avoiding bubbles) with a calibrated pipet, and use new pipet tips.

[0070] Add Coupled Beads, Standards, and Samples and then: 1. Cover unused wells with sealing tape. 2. Prewet the filter plate. 3. Vortex the diluted coupled beads for 30 sec at medium speed. Pour the diluted coupled beads into a reagent reservoir and add 50 µl to each well. TIP: A multichannel pipet is highly recommended for ease of use and efficiency. 4. Wash the wells twice with the wash method of choice. 5. Gently vortex the diluted standards, blanks, samples, and controls (if applicable) for 1-3 sec. Add 50 µl diluted standard, control, or sample to each well, changing the pipet tip after every volume transfer. 6. Incubate on shaker at room temperature as specified in Table 7 below.

[0071] Prepare and Add Detection Antibodies.

[0072] One tube of detection antibodies is included with each kit. Instructions are provided for diluting the detection antibodies to a 1x concentration. 1. While the samples are incubating, use the Calculation Worksheet shown below to calculate the volume of detection antibodies and detection antibody diluent needed. Detection antibodies should be prepared 10-15 min before use. 2. Add the required volume of detection antibody diluent to a 15 ml tube. 3. Vortex the detection antibodies for 15-20 sec at medium speed, then perform a 30 sec spin to collect the entire volume at the bottom of the vial. 4. Pipet the required volume from each detection antibody tube into a 15 ml polypropylene tube. Each well of the assay requires either 2.5 µl detection antibody (10x) or 1.25 µl detection antibody (20x) adjusted to a final volume of 25 µl.

[0073] Refer to the example detection antibody calculations in Tables 8-11 beow. These calculations include a 25% excess to compensate for transfer loss.

[0074] Tables 8-11 summarize the volumes required to prepare 1x detection antibodies from a single 10x or 20x stock. Also shown are volumes to prepare 1x antibodies when mixing two 10x or two 20x stocks. For instructions on preparing 1x antibodies from two stocks at different concentrations (for example when mixing human diabetes (20x) with human group I assays (10x), refer to the Bio-Plex Pro diabetes instruction manual (bulletin #10010747). 5. After incubating the samples, slowly remove and discard the sealing tape. 6. Wash three times with the wash method of choice. 7. Vortex the diluted detection antibodies gently for 1-3 sec. Pour the diluted detection antibodies into a reagent reservoir and add 25 µl to each well using a multichannel pipet. 8. Cover the plate with a new sheet of sealing tape and seal the wells. Incubate on shaker at room temperature as specified in Table 12 below.

[0075] Prepare and Add Streptavidin-PE 1. While the detection antibodies are incubating, use the Calculation Worksheet shown below to calculate the volume of streptavidin-PE (100x) and assay buffer needed. Each well requires 0.5 µl streptavidin-PE (100x) adjusted to a final volume of 50 µl with assay buffer. Streptavidin-PE should be prepared 10 min before use. 2. Add the required volume of assay buffer to a 15 ml tube. 3. Vortex the streptavidin-PE tube for 15-20 sec at medium speed. Perform a 30 sec spin to collect the entire volume at the bottom of the vial. 4. Pipet the required volume of streptavidin-PE into a 15 ml polypropylene tube containing assay buffer to dilute the streptavidin-PE to a 1x concentration. Table 13 nelow shows an example calculation to dilute streptavidin-PE, which includes a 25% excess to compensate for transfer loss. Protect the streptavidin-PE from light until ready to use. 5. After detection antibody incubation, slowly remove and discard the sealing tape. 6. Wash three times with the wash method of choice. 7. Vortex the diluted streptavidin-PE at medium speed for 3-5 sec. Pour the diluted streptavidin-PE into a reagent reservoir and add 50 µl to each well using a multichannel pipet. 8. Incubate on shaker at room temperature for the specified time shown in Table 14 below. 9. After the streptavidin-PE incubation step, slowly remove and discard the sealing tape. 10. Wash the wells three times with the wash method of choice. 11. Add 125 µl assay buffer to each well. Cover the plate with a new sheet of sealing tape. Shake the plate at room temperature at 1,100 rpm for 30 sec and slowly remove the sealing tape. Ensure that the plate cover has been removed before placing the plate on the reader. Reading the assay plate

[0076] Assay plates were read in accordance with the Instruction Manual, as described below.

[0077] Bio-Plex Manager ™< software is recommended for all Bio-Plex Pro assay data acquisition and analysis. Instructions for Luminex xPONENT software are also included. For instructions using other xMAP system software packages, contact Bio-Rad Technical Support or your regional Bio-Rad field applications specialist.

[0078] The protocol should be prepared in advance so that the plate is read as soon as the experiment is complete. A protocol file specifies the analytes used in the reading, the plate wells to be read, sample information, the values of standards and controls, and instrument settings.

[0079] Protocols may be obtained from within Bio-Plex Manager software version 6.0 or created from the File menu. Bio-Plex Manager software version 6.0 contains protocols for most Bio-Plex assays. The protocols should be chosen of new protocols should be created.

[0080] Protocols are prepared via the following steps: 1. Describe protocol and enter information about the assay. 2. Select analytes (from Table 2 above). 3. Format the plate according to the Plate Layout template created for the assay. 4. Enter details of the standards - e.g. highest concentration of each analyte, dilution factors, lot numbers, etc. 5. Enter controls information, including concentration and dilution information for each user-specified control for each assay. 6. Enter sample information, including the appropriate dilution factor. 7. Run the software protocols appropriate for the analytes concerned.

[0081] Data is acquired via the following steps: 1. Shaking the assay plate at 1,100 rpm for 30 sec, and visually inspecting plate to eensure that the assay wells are filled with buffer. 2. Run the protocol to start acquiring data. 3. Use the "wash between plates" function after each plate run to reduce clogging.

[0082] Data analysis and outlier removal is then performed.

[0083] Outliers are identified as standard data points that do not meet accuracy or precision requirements and should be considered invalid when performing curve fitting. As such, they should be removed to generate a more realistic and accurate standard curve. This may result in an extended assay working range and allow quantitation of samples that might otherwise be considered out of range (OOR).

[0084] In Bio-Plex Manager software version 6.0, outliers can be automatically removed by selecting the Optimize button in the Standard Curve window. In Bio-Plex Manager software 6.0 and earlier versions, outliers also can be manually selected in the Report Table.Calculations

[0085] The Bio-Plex ™< Pro instruction manual details the following calculations:Plan Plate Layout

[0086] 1. Fill out the 96-well plate template (page 43) as instructed in the Plan Plate Layout section (page 13). If using either a premixed panel or one singleplex assay, follow these directions. Enter the number of wells that will be used in the assay: _ (1) Calculations for Coupled Beads

[0087] 1. Determine the volume of 1x coupled beads needed. a. Each well requires 50 µl of coupled beads (1x): _ (1) x 50 µl = _ µl (2) b. Include a 20% excess to ensure enough volume: µl (2) x 0.20 = _ µl (3) c. Total volume of 1x coupled beads: µl (2) + _ µl (3) = _ µl (4) d. Volume of 20x coupled beads stock: µl (4) / 20 = _ µl (5) e. Volume of assay buffer required: _ µl (4) - _ µl (5) = _ (6) Calculations for Coupled Beads

[0088] 1. Determine the volume of 1x coupled beads needed. a. Each well requires 50 µl of coupled beads (1x): _ (1) x 50 µl = _ µl (2) b. Include a 20% excess to ensure enough volume: µl (2) x 0.20 = _ µl (3) c. Total volume of 1x coupled beads: µl (2) + _ µl (3) = _ µl (4) d. Volume of 20x coupled beads stock: µl (4) / 20 = _ µl (5) e. Volume of assay buffer required: _ µl (4) - _ µl (5) = _ (6) Calculations for Coupled Beads

[0089] 1. Determine the volume of 1x coupled beads needed. a. Each well requires 50 µl of coupled beads (1x): _ (1) x 50 µl = _ µl (2) b. Include a 20% excess to ensure enough volume: µl (2) x 0.20 = _ µl (3) c. Total volume of 1x coupled beads: µl (2) + _ µl (3) = _ µl (4) d. Volume of 20x coupled beads stock: µl (4) / 20 = _ µl (5) e. Volume of assay buffer required: _ µl (4) - _ µl (5) = _ (6) If mixing singleplex assays, follow these directions.Calculations for Coupled Beads

[0090] 1. Determine the volume of 1x coupled beads needed. a. Each well requires 50 µl coupled beads (1x): _ (1) x 50 µl = _ µl (2) b. Include 20% excess to ensure enough volume: _ µl (2) x 0.20 = _ µl (3) c. Total volume of 1x coupled beads: _ µl (2) + _ µl (3) = _ µl (4) d. Enter the number of diabetes single set (or analytes) tubes that will be multiplexed = _ (5) e. Volume of 20x coupled beads required from each coupled beads tube: _ µl (4) / 20 = _ µl (6) f. Total volume of diabetes bead stock required: (5) x _ µl (6) = _ µl (7) g. Volume of assay buffer required: _ µl (4) - _ µl (7) = _ µl (8) Calculations for Detection Antibodies

[0091] 2. Determine the volume of 1x detection antibody needed. a. Each well requires 25 µl detection antibodies (1x): _ (1) x 25 µl = _ µl (9) b. Include a 25% excess to ensure enough volume: _ µl (9) x 0.25 = _ µl (10) c. Total volume of 1x detection antibodies: _ µl (9) + _ µl (10) = _ µl (11) d. Enter the number of diabetes single set (or analytes) tubes that will be multiplexed = _ (5) e. Volume of 20x detection antibodies required from each detection antibody tube: _ µl (11) / 20 = _ µl (12) f. Total volume of diabetes detection antibody stock: _ µl (12) x _ (5) = _ µl (13) g. Volume of detection antibody diluent required: _ µl (11) - _ µl (13) = _µl (14) Calculations for Streptavidin-PE

[0092] 3. Determine the volume of 1x streptavidin-PE needed. a. Each well requires 50 µl streptavidin-PE (1x): _ (1) x 50 µl = _ µl (15) b. Include 25% excess to ensure enough volume: _ µl (15) x 0.25 = _ µl (16) c. Total volume of 100x streptavidin-PE: _ µl (15) + _ µl (16) = _ µl (17) d. Volume of 100x streptavidin-PE required: _ µl (17) / 100 = _ µl (18) e. Volume of assay buffer required: _ µl (17) _ µl (18) = _ µl (19) Processing of Data

[0093] The blood plasma samples from the 50 glioma patients and 27 healthy subjects were all assayed against various cytokines and angiogensis factors, and the levels of said cytokines and angiogensis factors determined in each case. A mean value for the cytokine and angiogensis factor levels for the 50 glioma patients ("Glioma Mean") and a mean value for the cytokine and angiogensis factor levels for the 27 healthy subjects ("Control Mean") was produced for each respective cytokine and angiogensis factor that was assayed, and the results compared. A statistical comparison was then made as to the significance of the particular cytokine and angiogensis factor in relation to its capacity to indicate the presence of gliomas.

[0094] Figures 1 to 7 show a graphical representation of the "control mean" (light grey) and "glioma mean" (dark grey), and also error bars, in relation to IL-8, Angiopoietin, Follistatin, HGF, Leptin, PDGF-BB, and PECAM-1 respectively.

[0095] Figures 7A to 7F show graphical representations of the "control mean" (dark grey - left), "low grade glioma mean" (light grey - middle), and "high grade glioma mean" (medium grey - right) and also error bars, in relation to FGF, G-CSF, sHER2neu, sIL-6Ralpha, Prolactin, and sVEGFR1 respectively. These figures demonstrate the applicability of the present invention to both low and high grade gliomas.

[0096] Figure 8 is a scatter-graphical correlation chart for PECAM-1 and PDGF-BB showing the relationship between PECAM-1 and PDGF-BB levels in the 50 glioma patients, and demonstrating a degree of linearity and a correlation coefficient of 0.45. This suggests that considering the relative levels of both PECAM-1 and PDGF-BB may provide a good correlation with a favourable or unfavourable diagnosis in relation to glioma.Results

[0097] Table 15 below compares the "control mean" concentrations of each assayed cytokine and angiogensis factor with the "glioma mean" concentrations of each assayed cytokine and angiogensis factor, and reports the "significance" of the particular cytokine or angiogensis factor in question (i.e. whether or not said cytokine or angiogensis factor is a suitable biomarker in blood plasma for glioma). Table 15 - Comparison of "Control Mean" and "Glioma Mean" to determine Significance as a Biomarker for Glioma Cytokine / Angiogenesis factor Control mean pg / ml Glioma mean pg / ml Significance (P<0.05) IL-2 2.0604347832.113NoIL-4 0.2040.225NoIL-6 3.7306252.6252NoIL-10 3.1134785.4772NoGM-CSF 00.8078NoIFN-γ 7.7973911.93551YESTNF-α 8.538260878.5034NoAngiopoietin 282.3258195.1382YESFollistatin 407.9671757.4796YESHGF 915.65831073.045YESIL-8 20.1334816.37449YESLeptin 5452.4019102.635YESPDGF-BB 2817.7924866.84YESPECAM-1 2734.1373832.264YESVEGF 64.2916769.3364NoFGF 184.6220.2YesG-CSF 379.4438.4YessHER2neu 4845.33604.8YesslL-6Ralpha 9603.112672.6YesProlactin 8101.927827.7YessVEGFR1 736.6911YesPDGF AA 73158578Yes

[0098] As will be apparent, at least IFN-γ, Angiopoietin, Follistatin, HGF, IL-8, Leptin, PDGF-BB, PECAM-1, PDGF-AA, sHER2 neu, sIL-6R alpha, prolactin, sVEGFR1, G-CSF, and FGF show a high degree of "significance", though the significance of IFN-γ was treated with caution given that many individuals demonstrated a zero concentration of this particular cytokine. In addition, it was observed that levels of Follistatin are higher in glioma patients than healthy subjects, Interleukin 10 is higher, Angiopoetin is lower, Leptin is higher, and PDGF-BB are higher. These cytokines and angiogenesis factors are therefore clearly excellent candidates as blood plasma biomarkers of gliomas.

[0099] In view of the above disclosure, relevant diagnostic systems and methods can be readily developed, using routine workshop techniques known in the art.

[0100] The above data is further corroborated by immunohistochemical comparisons between glioma brain tissue and non-cancerous brain tissue. Figures 8A-8G shows photographic immunohistochemical comparisons between glioma and non-cancerous brain tissues, namely: a) glioma tumour section x40 magnification showing positively staining and non-staining tumour cells; b) glioma tumour section x40 magnification showing negatively staining blood vessels; c) non-cancerous brain tissue x40 magnification showing negatively staining blood vessel; d) glioma tumour section x40 magnification showing interstitial staining; e) glioma tumour section x40 magnification showing interstitial staining, particularly of axonal tracts; f) non-cancerous brain tissue x40 magnification showing negatively staining blood vessel; g) choroid plexus tissue showing positive cytoplasmic staining.

[0101] Figures 8A-8G show, in particular, immunohistochemical staining of Follistatin, thereby showing an increased accumulation of this protein in the brain tissue of glioma patients.

[0102] Figures 8A-8G show the ability of Follistatin to identify tumour margins during immunohistochemical staining of brain tissue. Some gliomas exhibited significant follistatin immunostaining of tumour cells, many appeared to express gemistocytic morphology. However staining was not uniform throughout the tumour sample and some cells were patently immuno-negative (Fig. a). Positive immunostaining was entirely cytoplasmic with no membrane or nuclear component and other tissue elements within the sections, including blood vessels, were completely negative (Fig. b). There were no specific features of the tumours or constituent cells which were evidently predictive of immunopositivity or to account for the significant variability between individual tumours. The non cancerous (viz. normal) brain tissue was uniformly negative throughout and there was no staining of either neurones or glial cells (Fig c). There was a distinct interstitial stain in the presence of negatively staining cells that followed the axonal tracts of the sections (figs. d and e). There was no specific axonal staining and some of the axonal tracts did not take up any stain. The non-cancerous brain axonal tracts were uniformly negative (fig. f). There was some specific cytoplasmic staining of some cells from the choroid plexus (fig. g). This may suggest that Follistatin is being secreted into the CSF.Abbreviations

[0103] Basic FGF - Basic fibroblast growth factor G-CSF - Granulocyte-colony stimul.factor GM-CSF - Granulocyte-macrophage colony stimulating factor HGF - Hematopoietic growth factors, ICAM-1 - Intercellular adhesion molec. 1 IFN-gamma - Interferon gamma IL-1α - Interleukin 1 alpha IL-1β - Interleukin 1 beta IL-1ra - Interleukin 1 receptor antagon. IL-1 R1 - Interleukin 1 receptor-rel.prot 1 IL-1 R4 / ST2 - Interleukin 1 receptor 4, ST2 IL-2 - Interleukin 2 sIL-2 Rα - Interleukin 2 soluble receptor α IL-4 - Interleukin 4 IL-6 - Interleukin 6 IL-6 R - Interleukin 6 receptor IL-8 - Interleukin 8 IL-10 - Interleukin 10 PDGF AA - Platelet-derived growth factor PDGF-BB - Platelet-derived growth factor sHER2 neu - Human Epidermal Growth Factor Receptor 2 sIL-6R alpha - soluble Interleukin-6 receptor alpha sVEGFR1 - soluble vascular endothelial growth factor receptor 1 TNF-α - Tumor necrosis factor-alpha TNF-β - Tumor necrosis factor-beta VEGF - Vascular endothelial growth f.

Claims

1. A method of diagnosing glioma in a subject, the method comprising assaying a blood plasma sample of the subject in respect of two or more biomarkers selected from the group consisting of Angiopoietin, Follistatin, HGF, IL-8, Leptin, PDGF-BB, PECAM-1, PDGF-AA, sHER2 neu, sIL-6R alpha, prolactin, sVEGFR1, G-CSF, and FGF, wherein one of the two or more biomarkers is prolactin; and correlating elevated or reduced levels of each of the two or more biomarkers, whether relative to a predetermined threshold or relative to each other, with a determination of the presence of glioma in the subject.

2. The method as claimed in claim 1, wherein the two or more biomarkers include follistatin.

3. The method as claimed in claim 1 or claim 2, wherein assaying the blood plasma sample of the subject is performed in respect of three or more biomarkers selected from any one of the groups of claims 1 to 2.

4. The method as claimed in any of claims 1 to 2, wherein the method comprises correlating elevated or reduced levels of each of the two or more biomarkers, relative to a predetermined threshold, with a determination of the presence of glioma in the subject.

5. The method as claimed in claim 1 or claim 4, wherein correlating the analytical results with a determination of the presence of glioma in the subject involves an initial comparison of the analytical results with a reference standard or with previous analytic results that have been pre-correlated with a determination of the presence of glioma in the subject.

6. The method of any of claims 1 to 5, wherein the blood plasma sample is assayed with an immunoassay.

7. The method of any of claims 1 to 6, wherein the blood plasma sample is assayed using a magnetic bead-based multiplex assay designed to measure multiple biomarkers.

8. The method of claim 7, wherein the assay employs a plurality of fluorescently dyed beads to simultaneously detect multiple biomarkers in a single assay.

9. The method according to any preceding claim, wherein the glioma is low grade glioma.

10. The method according to any of claims 1 to 8, wherein the glioma is high grade glioma.

11. A diagnostic system for diagnosing glioma in a subject, comprising: a device configured to receive a blood plasma sample from the subject and configured to assay the blood plasma sample in respect of the two or more biomarkers defined in any of claims 1 to 10; and a computer configured to correlate or facilitate correlation of the amounts of the two or more biomarkers within the blood plasma sample with a determination of the presence of glioma in the subject, wherein the computer is configured to carry out the step of correlating elevated or reduced levels of each of the two or more biomarkers, whether relative to a predetermined threshold or relative to each other, with a determination of the presence of glioma in the subject, as defined in the method of any of claims 1 to 10.

12. The diagnostic system of claim 11, wherein the device comprises the computer.