WO2009043115A1 - Method for identifying biomarkers which are diagnostic for a pathological condition - Google Patents

Abstract

The present invention is to a method and assay for identifying biological markers and particularly biological markers of a pathological condition from a non-keratinous sample of a subject. The biological markers are characterised as specific biomarkers for a particular pathological state based on experimental analysis involving the introduction of a cell culture from a pathological state into an animal model and subsequently analysing a keratinous sample of the animal for any change in the ultrastructure of the keratin using X-ray diffraction techniques. The biomarkers of the present invention alter the ultrastructure of keratin.

Description

"Method for identifying biomarkers which are diagnostic for a pathological condition"

Cross-Reference to Related Applications

The present application claims priority from Australian Provisional Patent Application No 2007905442 filed on 4 October 2007, the content of which is incorporated herein by reference.

Field of the invention

The present invention relates to a method for identifying biological markers and particularly biological markers of a pathological condition, for example, cancer.

Background Art

The early and accurate detection of cancer is key in many cases to a successful therapeutic outcome.

The present applicant previously described a method for detecting cancer, including breast cancer by analysing a hair of a patient. The analysis involved X-ray diffraction as described in US Patent No 6,718,007, the contents of which are herein incorporated. The hair fibre of a patient suffering from cancer was seen to differ in its ultrastructure from hair fibres from healthy patients

While successful in identifying the change in hair associated with cancer, other factors must be considered including the fact that a large number of individuals have chemically treated hair. Such treatments interfere with the hair analysis and may result in false positives and false negatives.

The present invention aims to provide an alternative method for identifying biological markers of a pathological condition which cause a change in the ultrastructure of hair using samples other than hair. Summary of the Invention

In a first aspect, the present invention provides a method for detecting a pathological state or a pre-disposition for a pathological state in a subject, said method including: obtaining a non-keratinous sample from the subject; and analysing said sample and detecting one or more biomarkers indicative of said pathological state or a pre-disposition to said pathological state; wherein said one or more biomarkers are selected from a group comprising biomarkers which alter the ultrastructure of a keratinous material.

In a second aspect, the present invention provides an assay for detecting a pathological state or a pre-disposition for a pathological state, said assay including: a detecting means to detect one or more biomarkers in a non-keratinous sample taken from a subject, said one or more biomarkers being indicative of said pathological state or a pre-disposition to said pathological state; wherein said one or more biomarkers detected in the sample are selected from a group comprising biomarkers which alter the ultrastructure of a keratinous material.

In a third aspect, the present invention provides a method for identifying one or more biological markers of a pathological condition, the method including exposing a keratinous sample of a non-human animal to fibre X-ray diffraction and detecting changes in ultrastructure of said at least one keratinous sample, wherein cellular material of said pathological condition has been previously introduced into said animal, the method further including analysing a bodily fluid sample of the animal to identify said one or more biological markers wherein said one or more biological markers comprise at least one keratin-altering component.

In a fourth aspect, the present invention provides an isolated biological marker of a pathological condition characterised in that said biological marker is identified by a method which includes exposing a keratinous sample of a non-human animal to fibre X-ray diffraction and detecting changes in ultrastructure of said keratinous sample, wherein cellular material of said pathological condition has been previously introduced into said animal, the method further including analysing a bodily fluid sample of the animal to identify said biological marker and wherein said biological marker comprises at least one keratin-altering component.

The pathological condition may be cancer. Particularly, the pathological condition may be a cancer that causes a change in the ultrastructure of animal hair or whisker.

The subject referred to in aspects 1 and 2 is typically a human subject.

The cancer may be a member of the adenocarcinoma family. The cancer may include, but is not limited to: cancer of the breast, colon, skin (melanoma), prostate, lung, cervix, pancreas, stomach, vagina, oesophagus, kidney, ovary, duodenum, small intestine, rectum, salivary gland, or cecum.

The one or more biological markers may comprise one or more molecules forming part of, or secreted from, a cell having the pathological condition. Where the pathological condition is cancer, the one or more biological markers may originate from the cancer cell. In this embodiment, the cancer cell may secrete the at least one biological marker both in vitro and in vivo. Alternatively, the one or more biological markers may comprise components arising from the interaction between cancer cells and the animal or human host. As such, the biological markers of this embodiment may only be present in vivo.

The one or more biological markers are typically biologically active such that they cause a change in the ultrastructure of the hair or whisker. Said biological markers may bind to hair follicles and induce an alteration in their function leading to an altered alpha-keratin intermediate filament structure. Said alteration may be detectable by the

X-ray diffraction step.

The animal may be a rodent. In particular, the animal may be a mouse.

The cellular material may comprise one or more cells derived from subjects with the pathological condition. Further, the cellular material may comprise extracts and/or derivatives of the one or more cells. The cellular material may be obtained from a cell line of the pathological condition. The cell line may be any suitable cell line for a particular pathological condition.

In an embodiment of the invention, the method is for identifying a biological marker of breast cancer. In this embodiment, the cell lines used in said method may include, but are not limited to: BT-20, BT-474, Hs 190T, Hs319T, Hs329T, Hs344T. Hs350T, Hs371T, Hs578T, Hs749T, Hs841T, Hs849T, Hs851T, Hs861T, Hs905T, Hs479T, MCF-7, MCF-IOA, MDA-MB-231, MDA-MB-361, MDA-MB-435, MDA- MB-468, SK-BR-3, T-47D, ZR-75-1, HCC 1008.

In a further embodiment of the invention, the method is for identifying a biological marker of prostate cancer. In this embodiment, the cell lines used in said method may include, but are not limited to: PC3, PC-3M-luc-C6, LNCaP-lc-M6, OPCT-I, OPCT-2, OPCT-3, M12, DU- 145.

In a further embodiment of the invention, the method is for identifying a biological marker of colon cancer. In this embodiment, the cell lines used in said method may include, but are not limited to CaCo2, LoVo, WiDr, COLO 320DM, COLO 320HSR, DLD-I, COLO 205, COLO 201, HCT-15, SW620 or Hs675T.

In another embodiment, the cancer is cervical cancer and the cell line C-4II.

The animal may be infused with a conditioned medium from said cell lines. Said infusion may be relatively rapid or relatively slow. Said infusion may be continuous over a period of time. Alternatively said infusion may be intermittent over a period of time. In one embodiment, the animal may be infused with the cellular material over a period of between 3 and 6 weeks. Typically, the animal is infused continuously over a period of 4 weeks.

Other methods for the introduction of the cellular material are envisaged and include injecting the animal with the said cell lines. The animal may be injected with human-derived cancer cells or cell lines (xenografts) or, alternatively with same species-derived cancer cells or cell lines (allografts). In the latter case, it is envisaged that the allografts will produce proteins detectable in the serum of the animal which are homologues of human proteins arising from human cancer. In one embodiment, the animal may be injected once daily. Alternatively, the animal may be injected twice or more daily. The animal may be injected over a period of between 3 and 6 weeks. Typically, the animal is injected over a period of 4 weeks.

In a further embodiment, the animal may be injected once, and the injected cells left to form a substantial tumor over time. Typically, the time will be a period of 4-8 weeks.

The X-ray used may be derived from synchrotron radiation or other monochromatic X-ray sources providing X-rays within the energy range of five to twenty-five keV.

Upon detecting a change in the ultrastructure of the hair or whisker, a further step of fractionating the cellular material to isolate the one or more hair/whisker altering components may be undertaken. This step includes a number of methods including, but not limited to, techniques such as column chromatography, mass spectrometry and electrophoretic separations.

Upon isolation of the hair/whisker altering components, i.e. the biological markers, the invention encompasses the identification of said components. This step includes a number of methods including, but not limited to, techniques such as column chromatography, mass spectrometry and electrophoretic separations.

Following identification, a detector for the biological markers may be constructed. For example, the detector may comprise specific probes. Such probes include monoclonal antibodies.

The method of the invention allows the identification of biomarkers of a pathological condition and ultimately the development of detectors such as specific biomarker-sensitive probes which may be used to test for said pathological condition.

The probes may be used to provide a specific assay for the diagnosis of a particular pathological condition. The assay may use a non-keratinous sample including body fluids. In one embodiment, the body fluid includes blood, plasma or saliva. Preferably, the assay uses serum. Brief Description of the Drawings

Figure 1 provides a schematic representation of the steps of the method to determine particular biomarkers indicative of a pathological state;

Figure 2 is a schematic representation of the X-ray analysis system used in the method for determining biomarkers indicative of the pathological state;

Figure 3 shows an X-ray diffraction image of mouse whiskers prior to the introduction of cellular material from a cancer cell line;

Figure 4 shows an X-ray diffraction image of mouse whiskers post-introduction of cellular material from a cancer cell line and shows the addition of a ring (arrowed);

Figure 5 shows the gels of test sera compared to control sera of Example 1, the images obtained from a sample labelled with Cy3;

Figure 6 is a subset of the gel for Control 1 in Example 1 showing spots significantly different (up-regulated or down-regulated by 2-fold or more) in test serum compared to Control 1;

Figure 7 is a subset of the gel for Control 2 in Example 1 showing spots significantly different (up-regulated or down-regulated by 2-fold or more) in test serum compared to Control 2;

Figure 8 is a subset of the gel for Control 3 in Example 1 showing spots significantly different (up-regulated or down-regulated by 2-fold or more) in test serum compared to Control;

Figure 9 is a gel showing spots that have a fold change of 1.5 or more between test sera and at least one of the control serum samples in Example 2;

Figure 10 is a gel showing spots that show a fold change of 1.5 or more between test and Control 1 sera samples in Example 2; Figure 11 is a gel showing spots that show a fold change of 1 ,5 or more between test and Control 2 sera samples in Example 2;

Figure 12 is a gel showing spots that show a fold change of 1.5 or more between test and Control 3 sera samples in Example 2; and

Figure 13 is a gel showing spots that show a fold change of 1.5 or more between test and Control 4 sera samples in Example 2.

Detailed Description of an Exemplary Embodiment of the Invention

At the microscopic level, a whisker, which is a specialised type of hair fibre, consists of a highly organised and repetitive structure. Alterations in the whisker structure resulting from interference with the alpha keratin fibres or incorporation of substances into the fibre results in an altered X-ray diffraction pattern. As previously demonstrated by the applicant, the ultrastructure of a hair may be altered by cancer, giving an altered alpha-keratin X-ray diffraction pattern. The present invention provides a method of identifying a biological marker of a pathological condition including cancer wherein the marker comprises a component that alters the ultrastructure of a whisker of an animal. This is achieved by introducing cellular material of the pathological condition into an animal and particularly a mouse. X-ray diffraction analysis of a whisker is then undertaken. An altered whisker X-ray diffraction pattern will indicate presence of the pathological condition. Subsequent analysis including proteomic analysis of the cellular material introduced into the animal provides a method of isolating and subsequently identifying the whisker-altering components, that is, the biological markers of the pathological condition.

While a whisker of a mouse has been used in the experiments detailed herein, it is envisaged that other keratinous structures such as body hair and claws or nails could be used. Methodology

Cell Culture

Breast, prostate and colon cancer cell lines were purchased from the American Type Culture Collection (ATCC), Rockville, MD and cultured in a humidified incubator at 37⁰C and 5% CO₂ in tissue culture T-75cm² flasks, in appropriate growth medium.

Approximately 3OxIO⁶ cells were seeded individually into six 175cm² tissue culture flasks per cell line. After 2 days, the media was discarded and the cells rinsed twice with IX phosphate buffered saline (PBS). Following this, 30ml of serum-free medium, supplemented with glutamine (8mM) was added and the flasks incubated for an additional 24 hours. The conditioned media (CM) was collected and centrifuged at 900 rpm for 5 minutes to remove cellular debris. CM was then concentrated using urine concentrators or equivalent, and frozen at -8O⁰C until further use. A ImI aliquot was taken at the time of harvest to measure for total protein (Bradford assay).

The adhered cells were trypsinized and counted using a haemocytometer. In addition, 30ml of the serum-free medium was subjected to the same conditions as above, with no cells added, and used for comparison.

Mouse studies

Cells (10⁶) from a human breast cancer cell line MDA-MB-231 were injected subcutaneously on the back of nude mice and were left for 6 weeks to form significant tumors. Blood (from the tail vein) and whiskers were collected prior to injection and at termination. Whiskers were cut off as close to the skin of the snout as possible The serum fraction was collected from the blood by centrifugation, and stored at -8O⁰C until analysis.

In a further embodiment, concentrated conditioned medium (CCM) from each cell line was filtered to ensure sterility, then infused into mice continuously for 4 weeks, using an ALZET infusion pump. ALZET pumps were used for systemic administration when implanted subcutaneously or intraperitoneally. The pumps had the advantage that they could be attached to a catheter for intravenous infusion and enabled compounds of any molecular conformation to be delivered predictably at controlled rates, independent of their physical and chemical properties.

The conditioned medium was infused for four weeks. Whiskers were cut off as close to the skin of the snout as possible prior to commencement of infusion, at cessation of infusion, and four weeks after infusion has ceased.

In a further embodiment of the method, the mice were injected with the CCM subcutaneously (sc) rather than infused using the abovementioned pump.

X-ray diffraction studies

FIG. 3 depicts the system 1 for analysing hair or whiskers according to the present invention. An X-ray source, represented schematically by the arrow 2 is a collimated monochromatic X-ray beam which irradiates a single strand of whisker 3.

Reference numeral 4 refers to what is known in the art as the "evacuated X-ray flight path". Within this flight path, the scattered X-rays 5 are deflected from the direction of the unscattered beam 6. The unscattered beam 6 is occluded by a beam stop 7 while the scattered X-rays arrive at imaging plate 8 and are detected as shown schematically by reference numerals 9a, 9b, 9c and 9d.

X-ray fiber diffraction patterns for whiskers were collected using a monochromatic X-ray source such as a low-angle synchrotron facility for example, Sector 31, at the Advanced Photon Source (Argonne, IL, USA) with an X-ray wavelength ranging between 0.06 and 0.20 nm.

A single whisker was mounted on a holder which allowed the whisker to be aligned parallel to the long axis of the beam spot. The whisker was exposed to sufficient X-ray flux to generate a low angle diffraction pattern of alpha-keratin. Exposure time for the whisker to the incident beam was typically between 5 seconds and 5 minutes depending upon the flux of the incident beam. The exposure time was approximately 30 seconds on a third generation synchrotron source such as Sector 31. The X-ray patterns were recorded on an appropriate detector, for example a MAR 165 CCD detector. The space between the sample and the detector was evacuated so as to minimise air scattering, and was typically between 20 mm and 3000 mm. The analysis of the recorded patterns was carried out using two computer packages, SAXS 15ID and Fit2D.

Where there was evidence that the presence of cancer-derived material, either from implanted cancer cells or from CCM caused a change in the alpha-keratin pattern of the whiskers, proteomic methods were undertaken on the serum or the CCM in order to isolate the molecules responsible for the alteration.

Example 1

Test mice were injected subcutaneously with MDA-MB-231 cells in 200μl Matrigel. The cells formed vascularised tumors in the test mice over a 4-6 week period. Following X-ray diffraction studies as set out above, samples of serum were taken from control (pre-implanted mice and mice implanted with non-breast cancer- derived cellular material) and test mice for further analysis.

Serum was obtained from the mouse tail vein, before and after implantation of MDA-MB-231 cells and when the mice were euthanased. Albumin and IgG proteins were removed from the serum using immunoaffmity depletion, whereby these specific proteins were eluted using small recombinant immunoaffmity ligands.

The samples of sera tested were:

Control 1 : serum from mice prior to cell injection. Control 2: serum from mice injected with Matrigel only.

Control 3: serum from mice injected with MCF-10 A cells (human breast epithelial cells)

Test serum: serum from mice injected with MDA-MB-231 cells with Matrigel.

All serum samples were labelled with CyDyes and run on two-dimensional polyacrylamide gel electrophoresis. In the first dimension, isoelectric focusing, samples were separated based on their pi (isoelectric point). This was followed by the second dimension which was run perpendicular to the first and separated the samples based on their molecular weight. Approximately 100 μg of proteins of each sample was labelled with CyDye. The labelling protocol was followed according to the instructions provided with the CyDye kit. Each labelled protein sample was mixed according to the Table 1 and volume made up to 320 μl with sample buffer for each gel prior to reduction and alkylation of proteins. Proteins were reduced (5 mM TBP) and alkylated (10 mM acrylamide) for 60 min at RT after quenching the DIGE reaction. Reduced and alkylated samples were used for DIGE gels without storing the samples.

Table 1. DIGE experimental design

Gel # Cy 2 Cy 3 Cy 5

Gel 1 Pool (test and control 1 , 2 and 3) Test Control 1

Gel 2 Pool (test and control 1, 2 and 3) Test Control 2

Gel 3 Pool (test and control 1, 2 and 3) Test Control 3

Gel electrophoresis (1st and 2nd dimension)

First dimension: IPG strips pH 4-7 and 17 cm long (BioRad) were rehydrated with CyDyes labelled proteins. IEF was performed in the dark using the following protocol: 300 V for 4h, 1000 V for 2h, 3,000 V for 2h and 6000 V for 16h at 15⁰C. Immediately after the IEF step, IPG strips were equilibrated (equilibration buffer: 6 M urea, Ix Tris- HCl gel buffer pH 8.8, 3% (w/v) SDS, 50% (v/v) glycerol, 5mM TBP and 10 mM acrylamide) for 2x 15 min prior to the 2^nd dimension SDS-PAGE separation.

Second dimension: The SDS-PAGE 1.0 mm thick gels (6-18%T linear gradient) were poured in our laboratory using Tris-HCl buffer system. The equilibrated EPG strips were embedded on the top (lower gradient) of the 2nd dimension gels by 0.5% agarose and 0.001% Bromophenol Blue in Ix gel running buffer (cathode buffer). The second dimension gels were run on Protean II (BioRad) using a glycine cathode buffer (192 mM glycine, 0.1% SDS and 24.8 mM Tris base pH 8.3). The upper buffer contained additional 0.1% SDS (total 0.2% SDS). The gels were run overnight using the following program 5 mA/gel until 2 AM, 10 mA/gel until 9 AM and 50 mA/gel for Ih (or until the tracking dye Bromophenol Blue migrated from the gels) at 4⁰C under dark. The gel cassettes (gels remained in between glass plates) were rinsed with water and finally cleaned with 70% MeOH. The images were acquired one gel at a time. A variable mode laser scanner, Typhoon Trio (GE Healthcare), was used to acquire images of the gels using three different wavelengths. The emission filters used for acquisition of images were 520 nm for Cy2, 580 nm for Cy3 and 670 nm for Cy5. The PMT voltage was set to a point where the most abundant protein spots (2 to 3 spots) in a gel began to saturate while leaving areas of interest unsaturated. Gels were scanned at 100 μm resolution and the images were saved as 16-bit, gel files.

The gel images were uploaded into Progenesis Discovery 2005 (Nonlinear

Dynamics) as a 'Cross-Stain Analysis' experiment. Progenesis PG240, v.2006 (Nonlinear Dynamics) was used to complete the image analysis process. Spots were detected using auto spot detection method. For normalization of the data, Progenesis 'ratiometric' method for DIGE experiments was applied. All the statistical analysis was carried out from normalized data. Background was also subtracted from the images using Progenesis background subtraction method prior to quantitative analysis. The further step of manually editing the spots was carried out to delete streaks and remove background.

Spot Detection and Image Analysis

All spots detected in the multiplex images were added to the spot list of each physical gel (auto detected spots).

Following electrophoresis, gels were scanned using a laser scanner at different wavelengths and labelled protein spots were identified. Image analysis included protein spot detection, data normalization and statistical analysis.

An analysis of the gels to determine changes between the test and the controls, greater than 2-fold, was undertaken. The gels depicted in Figures 6, 7 and 8 show the spots which are either up-regulated or down regulated by greater than 2-fold. A summary of the differentially expressed proteins is provide below in Table 2.

Protein spots that were up-regulated in test mice in comparison to the control were chosen for further characterization and identification. Table. 2

The spots which show significantly changed up-regulation are then analysed to identify the proteins.

Example 2

In a further experiment, mouse sera was sampled as per the above methodology for Example 1, with the difference that the albumin and IgG were removed by the method outlined below:

The sera of Experiment 2:

Control 1: serum from mice prior to cell injection. Control 2: serum from mice injected with Matrigel only. Control 3: serum from mice injected with MCF-IO A cells Control 4: serum from mice injected with MDA-MB-231 cells without Matrigel Test serum: serum from mice injected with MDA-MB-231 cells with Matrigel.

Albumin and IgG depletion from Sera For each sample 50 μL of mouse sera was mixed with 50μL of equilibration buffer and added onto Sigma ProteoPrep® Immunoaffinity Albumin and IgG depletion spin columns according to the manufacturer's instructions. Each depleted sample was eluted in total of 225 μL of equilibration buffer. The bound samples containing the albumin and IgG protein species were eluted, diluted 1:2 with ultrapure water and stored at -80°C. A lOμL aliquot of each depleted and bound sample was run on a ID gel to evaluate the quality of the depletion step. The remaining sample was stored at - 8O⁰C until required.

Protein quantitation Protein concentrations of each sample were measured by Bradford (Sigma) assay.

Experimental design and CyDye labelling protocol for DIGE analysis

All the serum samples were labelled with CyDyes (minimal labelling) and paired for running gels as described in the table below. Approximately 150 μg of protein from each sample was labelled with CyDye. The labelling protocol was followed according to the manufacturer's instructions using 600 pmol of dye to label each 150μg aliquot of sample. Each labelled protein sample was mixed according to Table 3 and volume made up to 400 μL with sample buffer for rehydration.

Table 3. DIGE experimental design

st πd

Gel electrophoresis (1 and 2 dimension)

First dimension: IPG strips pH 4-7 and 17 cm long (BioRad) were rehydrated with CyDye labelled proteins. IEF was performed in the dark for a total of 90 kVh using the following protocol: lOOμA per strip for 100 V for 3h, 300 V for Ih gradient, 300 V for 3h, 1000 V for Ih gradient, 1000 V for 2h, 2500 V for Ih, 2500 v for Ih, 7500 V for 82.5 kVh at 20°C. After focusing, IPG strips were drained of oil and stored at -8O⁰C until required for 2^nd dimension separation.

Second dimension: Strips were defrosted, equilibrated for 15 mins at room temperature in a reducing buffer (6 M urea, Ix Tris-HCl gel buffer pH 8.8, 2% (w/v) SDS, 20% (v/v) glycerol, 65mM DTT) and then 15 mins in alkylating buffer (6M urea, Ix Tris-HCl gel buffer pH 8.8, 2% (w/v) SDS, 20% (v/v) glycerol, 2.5% acrylamide). The SDS-PAGE 1.0 mm thick gels (12% resolving gel, 4% stacking gel) were poured in our laboratory using Tris-HCl buffer system. The equilibrated IPG strips were embedded on the top of the 2nd dimension gels by 0.5% agarose and 0.001% Bromophenol Blue in Ix gel running buffer (cathode buffer). The second dimension gels were run using a Protean II system (BioRad) using a glycine cathode buffer (192 mM glycine, 0.1% SDS and 24.8 mM Tris base pH 8.3). The gels were run using the following program 10 mA/gel for Ih and then 40 mA/gel for 8-10 h (or until the tracking dye Bromophenol Blue migrated from the gels) at 10⁰C under dark.

Image acquisition

The gel cassettes (gels remained in between glass plates) were rinsed with water and finally cleaned with 70% MeOH. The images were acquired one gel at a time with a Molecular Imager Pharos® FX Plus laser scanner (Bio-Rad), using three different wave lengths. The emission filters used for acquisition of images were 530 nm for

Cy2, 605 nm for Cy3 and 695 nm for Cy5. The PMT voltage was set to a point where the most abundant protein spots (2 to 3 spots) in a gel began to saturate while leaving areas of interest unsaturated. Gels were scanned at 100 μm resolution and the images were saved as 16-bit.gel files.

Image analysis

The gel images were uploaded into Progenesis SameSpots v3 (Nonlinear Dynamics) as a 'Cross-Stain Analysis' experiment. SameSpots was used for the complete image analysis process whereby the gels were warped, spots were detected, the data was normalised for DIGE experiments and the background subtracted using the programs automated methods for each step. All statistical analysis was carried out from normalized data. Results

All samples labelled well with the fluorescent Cydyes and resolved well on 17cm pH 4-7, 12% 2-D gels.

Spots that exhibited a fold change of 1.5 or more were reported as the technical error associated with DIGE experiments in much lower than that experienced in standard 2-D gel electrophoresis. However, spots with a fold change of 2 or more should have more weight placed on them.

Table 4 and Figures 9-13 indicate the spots that show a fold change of 1.5 or more between proteins in the Test serum and at least one of the control serum samples.

The analysis conducted in Experiment 2 identifies different spots changing in abundance to the DIGE analysis run in Experiment 1. This may be due to the different serum depletion methods used. Several of the spots indicated to be changing in Experiment 1 are around the region where IgG (approximately pH 5.5-7.5, 28 kDa) would resolve and this protein is predominantly removed by the Sigma ProteoPrep® Immunoaffmity Albumin and IgG depletion columns used in Experiment 2

A summary of protein spots that were significantly upregulated in the test serum compared to the control sera is provided below in Table 4.

Table 4. - (The apparent molecular weights are based on the spots position in the gel in relation to a MW standard marker and the isoelectric point is calculated assuming the proteins are separating linearly in the IPG strips.)

(/)

C CO

(/)

m (a

I m m

Ti c T- m

N)

TJ O

Further processing of results.

Having analysed the gels obtained using the above methodology, certain candidate biomarkers are identified. Particularly, the proteins showing a marked up- regulation constitute candidate biomarkers of the pathological condition.

The candidate biomarkers are then identified using, in the present case, the technique of mass spectrometry. In this regard, the proteins are excised from the gel, eluted, run on a mass spectrometer and identified using analytical software.

The identified candidate biomarkers are then introduced into mice to determine/confirm which biomarker(s) actually cause a change in the ultrastructure of the whisker of a mouse. To determine if there had been a changed ultrastructure, the mice whiskers were subjected to X-ray diffraction analysis as set out above.

The whisker altering biomarkers identified in the above method are therefore indicative of a particular pathological state or a pre-disposition to that pathological state.

In the above specific examples, the pathological state being tested was breast cancer using the breast cancer cell line MDA-MB-231. In further studies, other breast cancer cell lines are tested for biomarkers using the above methodology. These cell lines include BT-20, BT-474, Hsl90T, Hs319T, Hs329T, Hs344T. Hs350T, Hs371T, Hs578T, Hs749T, Hs841T, Hs849T, Hs851T, Hs861T, Hs905T, Hs479T, MCF-7, MCF-IOA, MDA-MB-361, MDA-MB-435, MDA-MB-468, SK-BR-3, T-47D, ZR-75- 1.

Similarly, colon cancer cell lines are tested for biomarkers using the above methodology. The colon cancer cell lines include CaCo2, LoVo, WiDr, COLO 320DM, COLO 320HSR, DLD-I, COLO 205, COLO 201, HCT-15, SW620 or Hs675T.

Specific means to detect the biomarker proteins in specimens such as body fluids are then constructed. In the present case, the detecting means comprise monoclonal antibodies specific for the biomarker proteins. However, it is envisaged that other detection means may be utilised including the use of tagged peptides specific for a particular region of the biomarker or polyclonal antibodies.

Biological samples such as blood, plasma, saliva and serum are then screened for the biological markers using the detecting means.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

1. A method for detecting a pathological state or a pre-disposition for a pathological state in a subject, said method including: obtaining a non-keratinous sample from the subject; and analysing said sample and detecting one or more biomarkers indicative of said pathological state or a pre-disposition to said pathological state; wherein said one or more biomarkers are selected from a group comprising biomarkers which alter the ultrastructure of a keratinous material.

2. The method of claim 1 wherein the subject is a human subject.

3. The method of claim 1 or claim 2 wherein the pathological state is a cancer which alters the ultrastructure of a keratinous material.

4. The method of claim 3 wherein the cancer is selected from the one or more of the group comprising: cancer of the breast, colon, skin (melanoma), prostate, lung, cervix, pancreas, stomach, vagina, oesophagus, kidney, ovary, duodenum, small intestine, rectum, salivary gland, or cecum.

5. The method of any one of the preceding claims wherein prior to obtaining and analysing a non-keratinous sample, the one or more biomarkers have been identified as indicative of said pathological state by the method of introducing cellular material of said pathological condition into a non-human animal model, subsequently exposing a keratinous sample of said animal model to fibre X-ray diffraction and detecting any changes in ultrastructure of said sample, and wherein if changes are detected in the ultrastructure of the sample, further analysing a bodily fluid sample of the non-human animal to characterise candidates of said one or more biological markers.

6. The method of claim 5 wherein the cellular material is obtained from a cell line of the pathological state.

7. The method of claim 6 when dependent upon claim 4 wherein when the cancer is breast cancer the cell line is selected from one or more of BT-20, BT-474, Hsl90T,

Hs319T, Hs329T, Hs344T. Hs350T, Hs371T, Hs578T, Hs749T, Hs841T, Hs849T, Hs851T, Hs861T, Hs905T, Hs479T, MCF-7, MCF-IOA, MDA-MB-231, MDA-MB- 361, MDA-MB-435, MDA-MB-468, SK-BR-3, T-47D, ZR-75-1 or HCC 1008.

8. The method of claim 6 when dependent upon claim 4 wherein when the cancer is prostate cancer the cell line is selected from one or more of PC3, PC-3M-luc-C6,

LNCaP-lc-Mό, OPCT-I, OPCT-2, OPCT-3, M12 or DU- 145.

9. The method of claim 6 when dependent upon claim 4 wherein when the cancer is colon cancer the cell line is selected from one or more of CaCo2, LoVo, WiDr, COLO 320DM, COLO 320HSR, DLD-I, COLO 205, COLO 201, HCT- 15, SW620 or Hs675T.

10. The method of any one of claims 5 to 9 wherein following characterisation of said candidates, each candidate biomarker is introduced into a non-human animal model and a keratinous sample subsequently taken from said animal and analysed using X-ray diffraction to determine if said candidate biomarker alters the ultrastructure of the keratin.

11. The method of claim 10 wherein more than one candidate biomarker is introduced into a single animal.

12. The method of any one of claims 5 to 11 wherein once the one or more biomarkers are characterised, a detecting means, specific for said one or more biomarkers is constructed.

13. The method of claim 12 wherein the detecting means comprises at least one monoclonal antibody.

14. The method of any one of the preceding claims wherein the non-keratinous sample comprises a body fluid selected from the group comprising blood or a blood product including plasma and serum, or saliva.

15. An assay for detecting a pathological state or a pre-disposition for a pathological state, said assay including: a detecting means to detect one or more biomarkers in a non-keratinous sample taken from a subject, said one or more biomarkers being indicative of said pathological state or a pre-disposition to said pathological state; wherein said one or more biomarkers detected in the sample are selected from a group comprising biomarkers which alter the ultrastructure of a keratinous material.

16. The assay of claim 15 wherein the subject is a human subject.

17. The assay of claim 15 or claim 16 wherein the pathological state is a cancer which alters the ultrastructure of a keratinous material.

18. The assay of claim 17 wherein the cancer is selected from the one or more of the group comprising: cancer of the breast, colon, skin (melanoma), prostate, lung, cervix, pancreas, stomach, vagina, oesophagus, kidney, ovary, duodenum, small intestine, rectum, salivary gland, or cecum.

19. The assay of any one of claims 15 to 18 wherein prior to detecting one or more biomarkers from the non-keratinous sample, the one or more biomarkers have been identified as indicative of said pathological state by the method of introducing cellular material of said pathological condition into a non-human animal model, subsequently exposing a keratinous sample of said animal model to fibre X-ray diffraction and detecting any changes in ultrastructure of said sample, and wherein if changes are detected in the ultrastructure of the sample, further analysing a bodily fluid sample of the non-human animal to characterise candidates of said one or more biological markers.

20. The assay of claim 19 wherein the cellular material is obtained from a cell line of the pathological state.

21. The assay of claim 20 when dependent upon claim 18 wherein when the cancer is breast cancer the cell line is selected from one or more of BT-20, BT-474, Hs 190T, Hs319T, Hs329T, Hs344T. Hs350T, Hs371T, Hs578T, Hs749T, Hs841T, Hs849T, Hs851T, Hs861T, Hs905T, Hs479T, MCF-7, MCF-IOA, MDA-MB-231, MDA-MB- 361, MDA-MB-435, MDA-MB-468, SK-BR-3, T-47D, ZR-75-1 or HCC 1008.

22. The assay of claim 20 when dependent upon claim 18 wherein when the cancer is prostate cancer the cell line is selected from one or more of PC3, PC-3M-luc-C6, LNCaP-lc-Mό, OPCT-I, OPCT-2, OPCT-3, M12 or DU-145.

23. The assay of claim 20 when dependent upon claim 18 wherein when the cancer is colon cancer the cell line is selected from one or more of CaCo2, LoVo, WiDr, COLO 320DM, COLO 320HSR, DLD-I, COLO 205, COLO 201, HCT-15, SW620 or Hs675T.

24. The assay of any one of claims 15 to 23 wherein following characterisation of said candidates, each candidate biomarker is introduced into a non-human animal model and a keratinous sample subsequently taken from said animal and analysed using X-ray diffraction to determine if said candidate biomarker alters the ultrastructure of the keratin.

25. The assay of claim 24 wherein more than one candidate biomarker is introduced into a single animal.

26. The assay of any one of claims 15 to 25 wherein the detecting means comprises at least one monoclonal antibody.

27. The assay of any one of claims 15 to 26 wherein the non-keratinous sample comprises a body fluid selected from the group comprising blood or a blood product including plasma and serum, or saliva,