Patent Description:
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.

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.

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.

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.

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. com/webroot/web/pdf/lsr/literature/<NUM>. 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.

<NPL>, relates to investigations of eight circulating plasma markers in brain tumor patients.

<NPL>, relates to identification of blood protein biomarkers in the clinical assessment of malignant glioma.

<CIT> relates to methods of diagnosing higher- and lower-grade Astrocytoma using biomarkers.

<NPL>, relates to follistatin serum concentrations during full-term labour in women, and the variations thereof between spontaneous and induced labour.

<NPL>, relates to potential serum biomarkers of glioblastoma.

<NPL>, Retrieved from the Internet <URL:http://imr. com/content/<NUM>/<NUM>/<NUM>. pdf> [retrieved on <NUM>], relates to the expression of activin A and follistatin in glioblastoma and their effects on U87 in vitro.

<CIT> relates to methods for detecting a target nucleic acid and a target protein in a single assay.

<NPL>, Retrieved from the Internet <URL:https://www. gov/pmc/articles/PMC2268985/pdf/nihms-<NUM>. pdf> [retrieved on <NUM>], relates to a mediator of angiogenesis.

<NPL>, relates to conceptual and methodological issues relevant to cytokine and inflammatory marker measurements in clinical research.

<NPL>, relates to ELISA and multiplex technologies for cytokine measurement in inflammation and aging research.

<NPL>, relates to ISPUB considerations for the use of blood plasma and serum for proteomic analysis.

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.

According to an aspect, the present invention provides a method of diagnosing glioma in a subject as set forth in appended claim <NUM>. 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-<NUM>, Leptin, PDGF-BB, PECAM-<NUM>, 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.

According to an aspect, the present invention provides a diagnostic system for diagnosing glioma in a subject as set forth in appended claim <NUM>. 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.

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

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

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 <NUM>% of total blood volume. It is the intravascular fluid part of extracellular fluid (all body fluid outside of cells). It is mostly water (<NUM>% by volume) and contains dissolved proteins (major proteins are fibrinogens, globulins and albumins), glucose, clotting factors, mineral ions (Na+, Ca++, Mg++, HCO<NUM>- 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.

"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.

"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.

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

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..

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

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.

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.

In a particular embodiment, the glioma is a Grade III or Grade IV glioma.

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..

The subject suitably has or is suspected as having glioma.

The subject is suitably a glioblastoma or a gliosarcoma patient. In a particular embodiment, the subject is a glioblastoma patient.

In the context of the methods and diagnostic systems of the present invention, a blood plasma sample is used.

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-<NUM>, Leptin, PDGF-BB, PECAM-<NUM>, 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.

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.

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 the blood plasma sample suitably involves determining the levels of the two or more biomarkers within the blood plasma sample.

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.

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

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.

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 <NUM> hrs, using as little as <NUM>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.

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 <NUM> unique fluorescently dyed beads are used for cytokine/angiogenesis factor detection.

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.

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

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.

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.

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).

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 <NUM>. 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.

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).

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. com, and in particular at http://www. com/webroot/web/pdf/lsr/literature/<NUM>. 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 <NUM>-well Bio-Plex Pro flat-bottom plates were washed via magnetic separation using the magnetic setting of the Bio-Plex Pro wash station. The <NUM>-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.

As described in the Instruction Manual, The Bio-Plex™ suspension array system is built around the three core elements of xMAP technology:.

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 (<FIG>). 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.

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 <NUM> reader for example, a red (<NUM>) laser illuminates the_fluorescent dyes within each bead to provide bead classification and_thus assay identification. At the same time, a green (<NUM>) 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.

The Instruction Manual summarises the initial preparation for the assays as follows:.

The Instruction Manual summarises the running of the assays as follows:.

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 <NUM>):
<IMG>.

In accordance with the Instruction Manual, the testable cytokines include (Table <NUM>):
<IMG>.

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 samples were collected from <NUM> glioma patients and <NUM> healthy subjects.

Blood plasma samples for each of the <NUM> glioma patients and <NUM> healthy subjects were prepared by adding the corresponding fresh whole blood sample to a tube containing an anticoagulant, and spinning the tube at <NUM>,<NUM> rpm for <NUM> at <NUM> 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 <NUM>/m<NUM>, or <NUM>/l. The blood plasma samples were then either assayed immediately or otherwise aliquoted and stored in single use aliquots at -<NUM> for later use, though repeated freeze/thaw cycles were avoided.

Before conducting the assays, <NUM> volume of plasma sample was diluted with <NUM> volumes of sample diluents (for example, <NUM>µL sample + <NUM>µL sample diluents).

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

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

When using <NUM>-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 <NUM> reservoir.

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

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.

Add Coupled Beads, Standards, and Samples and then:.

One tube of detection antibodies is included with each kit. Instructions are provided for diluting the detection antibodies to a 1x concentration.

Refer to the example detection antibody calculations in Tables <NUM>-<NUM> beow. These calculations include a <NUM>% excess to compensate for transfer loss.

Tables <NUM>-<NUM> 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 #<NUM>).

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

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.

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.

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

Protocols are prepared via the following steps:.

Data is acquired via the following steps:.

Data analysis and outlier removal is then performed.

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).

In Bio-Plex Manager software version <NUM>, outliers can be automatically removed by selecting the Optimize button in the Standard Curve window. In Bio-Plex Manager software <NUM> and earlier versions, outliers also can be manually selected in the Report Table.

The Bio-Plex™ Pro instruction manual details the following calculations:.

If mixing singleplex assays, follow these directions.

Determine the volume of 1x detection antibody needed.

Determine the volume of 1x streptavidin-PE needed.

The blood plasma samples from the <NUM> glioma patients and <NUM> 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 <NUM> glioma patients ("Glioma Mean") and a mean value for the cytokine and angiogensis factor levels for the <NUM> 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.

<FIG> show a graphical representation of the "control mean" (light grey) and "glioma mean" (dark grey), and also error bars, in relation to IL-<NUM>, Angiopoietin, Follistatin, HGF, Leptin, PDGF-BB, and PECAM-<NUM> respectively.

<FIG> 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.

<FIG> is a scatter-graphical correlation chart for PECAM-<NUM> and PDGF-BB showing the relationship between PECAM-<NUM> and PDGF-BB levels in the <NUM> glioma patients, and demonstrating a degree of linearity and a correlation coefficient of <NUM>. This suggests that considering the relative levels of both PECAM-<NUM> and PDGF-BB may provide a good correlation with a favourable or unfavourable diagnosis in relation to glioma.

Table <NUM> 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).

As will be apparent, at least IFN-γ, Angiopoietin, Follistatin, HGF, IL-<NUM>, Leptin, PDGF-BB, PECAM-<NUM>, 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 <NUM> 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.

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

The above data is further corroborated by immunohistochemical comparisons between glioma brain tissue and non-cancerous brain tissue. <FIG> 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.

<FIG> show, in particular, immunohistochemical staining of Follistatin, thereby showing an increased accumulation of this protein in the brain tissue of glioma patients.

Claim 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-<NUM>, Leptin, PDGF-BB, PECAM-<NUM>, 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.