"Enhanced Method of Detection"
Cross-Reference to Related Applications The present application claims priority from Australian: Provisional Patent
Application No 2009902727 filed on 12 June 2009, the content of which is incorporated herein by reference.
Field of the Invention
The present invention relates to diagnostics and enhancing diagnostic tools and methods to identify one or more conditions in a subject.
Background Art
Methods for early stage detection of disease in order to maximize treatment outcomes are clearly desirable. Cancer, and in particular breast cancer, is one example that clearly demonstrates excellent survival statistics when early-stage disease is treated using current therapies. However, for many patients diagnosis is made too late. If all cases of breast cancer could be detected prior to metastasis then there would be a significant reduction of both individual mortality and the economic burden on the community. One of the key imperatives in cancer research, including breast cancer research, therefore, is the need to develop more effective screening tools for the early detection of disease.
Biomarke.rs are biological molecules that are indicators of biological and pathological processes, or physiological and pharmacological responses to a drug treatment. Since biomarkers can be used to measure the progress of disease or the response to treatment, they have very significant potential roles to play in both the diagnosis and prognosis of disease. Ideally, in the case of breast cancer a biomarker signature would be able to detect cancer in asymptomatic patients and improve the accuracy of screening mammograms. A reliable biomarker signature may also signify new cancer,, even in the setting of normal physical examination results, and would indicate further more intensive diagnostic workup and/or preventive treatment.
■ . Reported differences in the small angle X-ray scattering (SAXS) patterns of hair from individuals with breast cancer compared to healthy subjects has been reported and is the subject of WO 00/34774, the contents of which are herein incorporated by reference.
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The SAXS patterns of hair from cancer patients contained a ring of comparatively low intensity which was superimposed on the normal α-keratin pattern obtained from healthy control subjects. The nature of the ring seen on the X-ray diffraction (XRD) pattern to date has been unknown.
The present invention, based on an understanding of the underlying molecular cause of the ring provides an improved method to identify conditions such as breast cancer. This understanding and the findings set out in this invention also allow identification of conditions other than breast cancer,
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.
Summary of the Invention A method of enhancing an X-ray diffraction image of an abnormal keratin- containing sample obtained from a subject wherein the identification of an abnormal keratin-containing sample is indicative of one or more conditions, said method including:
obtaining a keratin-containing sample from the subject;
applying an enhancer agent to said sample;
subjecting the sample to X-rays to obtain said X-ray diffraction image;
wherein -the enhancer agent enhances a change present in an abnormal keratin- containing sample, said change having been caused by said condition. A method of identifying a condition which changes a lipid profile in a keratin- containing component of a subject, said method including: obtaining a sample of said keratin-containing component from the subject;
applying an enhancer agent to said sample;
subjecting the sample to X-rays to obtain said X-ray diffraction image;
wherein the enhancer agent enhances a change present in the sample from a subject having said condition, said change resulting from a changed lipid profile in said keratin-containing component.
The method of either of the above aspects wherein said condition includes any condition which results in an increase in lipid in keratin. The condition may comprise a neoplastic condition. The neoplastic condition may comprise a wide range of cancers and is not limited to a specific cancer, with the proviso that. said cancer alters the lipid profile of the keratin. Examples of cancers include cancer of the breast, colon, lung, cervix, pancreas, stomach, vagina, oesophagus, kidney, ovary, duodenum, small intestine, rectum, salivary gland, or cecum.
The condition may result in an alteration of the lipid profile in the keratin- containing component by a number of pathways. The condition may cause an alteration in normal metabolic pathways of one or more lipid species. The alteration in the keratin-containing component which may be detected by X- ' ray diffraction analysis may result from the incorporation or altered biosynthesis of one . or more of a large number of lipids in the sample taken from the subject. Examples include, but are not limited to phospholipids including phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphocholine; sphingolipids including sphingomyelin, sphingosine; palmitic acid, palmitoleic acid, arachidonic acid; linoleic acid and choline.
In one embodiment, the condition may alter normal phospholipid metabolism or composition in body tissues and/or fluids. In this embodiment, various metabolites in the phospholipid metabolism, pathway may be altered. As an example, the metabolic pathway of choline may be altered to ultimately cause an altered composition or biosynthesis of choline phospholipids in a keratin-containing component.
In a further embodiment, the condition may cause a change in the profile of a fatty acid containing compound such as sebum in a keratin-containing component of the subject. The keratin-containing component of the present invention may include, but is not limited to any type of body hair, nail, skin and cuticle. The keratin-containing sample may be taken from any of these components..
The enhancer agent may be any agent which reacts with lipids. The ieaction with the lipid provides an enhanced X ray diffraction pattern of the lipid feature.
The enhancer agent may react with specific groups on lipids. For example, the enhancer agent may react with phosphate groups on certain lipids. Further, the enhancer agent may include an imidazole group which reacts with the phosphate group on the phospholipids. In a. further embodiment, the enhancer agent preferably includes a metal group. The metal group preferably has a high electronic density to generate a strong X-ray diffraction signal. Further examples of enhancer agents include alkaline salts. Still further, the enhancer agent may be lead nitrate or lead acetate.
According to another aspect of the present invention, there is provided a method for enhancing an X-ray diffraction image of an abnormal keratin-containing sample obtained from a subject wherein the identification of an abnormal keratin-containing• sample is indicative of one or more conditions, said method including:
subjecting the keratin-containing sample to an enhancer agent;
providing an X-ray source producing a beam of X-ray radiation;
positioning said keratin-containing sample on a sample stage within said beam; . detecting the scattering of said X-ray beam caused by said keratin-containing sample; and
displaying the output;
whereby a pattern of output indicative of an abnormal keratin-containing sample is enhanced by said enhancer agent. In one embodiment of any of the above aspects, the X-ray diffraction pattern of the keratin-containing material includes a ring at a certain spacing from the centre of the pattern. The enhancer agent of the present invention intensifies the X-ray diffraction of this ring such that the ring is more prevalent when analysed. A particularly weak signal, for example in instances where the lipid profile may have been only changed slightly will more likely be noted upon analysis due to enhancement by the enhancer agent. In the embodiment wherein the condition comprises breast cancer, the enhanced ring of the present invention may be observed at a molecular spacing of 4.76 ± 0.07 nm. (Corino G & French P, M J Cancer 122: 847-856, 2008)
If the lipid profile of. a keratin-containing sample is altered due to a subject suffering from a condition which alters this lipid profile, the use of a specific enhancer that reacts with lipid may provide a means to enhance the altered aspect of the X-ray diffraction pattern of the keratin-containing sample.
Brief Description of the Drawings
Figure 1 shows A) a normal X-ray diffraction pattern of hair and B) an abnormal X-ray diffraction pattern of hair;
Figure 2 shows A) an X-ray diffraction pattern of a hair with no ring and B) the X-ray diffraction pattern following soaking of the hair of A) in olive oil;
Figure 3 shows the effect of solvent on the X-ray diffraction pattern of a hair exhibiting a ring;
Figure 4 shows enhancement of a ring observed from the results of X-ray diffraction by using lead nitrate. Description of an Exemplary Embodiment of the Invention Examples
In the following described experimentation, the X-rays used are derived from synchrotron radiation or other monochromatic X-ray sources providing X-rays within the energy range of five to twenty-five keV.
Where the keratin-containing component studied was hair, a single hair was used. The hair was mounted on a holder under sufficient" tension to maintain alignment. The holder was mounted on a motorised translation device capable of moving in lμm steps in the vertical and horizontal planes which enabled each sample to be precisely located in the X-ray beam. The hair fibres were mounted "with the axis of the hair in the parallel plane and at a 90 degree angle of incidence to the X-ray source. The wavelength of the X-ray beam was approximately 1.1 A and had a resolution (ΔE/E) of lχl 0"4.
Each fibre was exposed to the X-ray source for a set period of time depending on the intensity of the beam. In general each sample was exposed to a total of approximately IxIO14 photons. The resultant diffraction patterns were collected on a MARl 65 CCD detector. Sample to detector distance was approximately 600 mm.
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Experiments
To determine that the ring found in a sample of hair from a breast cancer patient (as shown in Fig IB) is lipid-derived.
From the applicant's previous studies, it is evident that certain conditions such as breast cancer, colon cancer and Alzheimer's disease result in the alteration of a keratin- containing component such as hair. This alteration may be detected using the X-ray diffraction technique outlined above. In patients suffering from breast cancer, a ring is observed. The diffraction pattern from a sample of hair from a healthy person is shown in Fig. IA and that from a breast cancer patient shown in Figure IB. The ring reported to be associated with the presence of breast cancer can be seen in the diffraction pattern in Fig IB. To date, it has been unclear what causes this ring to appear. It has been speculated that it is due to an 'additional component' secreted from the breast cancer and taken up by the hair. The present invention addresses this by the finding that the ring is due to the presence of additional lipids incorporated into the structure of keratin- containing components.
Experiment 1
Addition of fatty acids to hair
A sample of hair which did not have a ring (this .was confirmed by X-ray diffraction) was selected. The diffraction pattern of this hair is shown in Figure 2A. The hair was then soaked in olive oil for 10 minutes, wiped dry and then re- exposed to X-rays. The resulting diffraction pattern is depicted in Figure 2B wherein a clear ring is visible. The ring resulting from soaking in olive oil is very similar in appearance and d-spacing to the ring shown in Figure IB from a breast cancer patient.
Experiment 2
Solvent extraction of hairs Hairs from breast cancer patients which displayed rings were run on a synchrotron SAXS beamline (Australian Synchrotron) to confirm the presence of the ring. The individual hairs were then soaked in a solvent for 2 hours, rinsed in MiIIiQ water, dried and then re-run very close to the same point on the hair fibre. The solvents used were:
A Isopropanol (100%)
B Chloroform (100%)
C Chloroform/methanol (1:1)
D Chloroform/methanol (1:2)
The results are shown in Fig 3.
* While all solvents reduced the intensity of the ring in most fibres, it was evident that consistently, isopropanol was the least effective and chloroform/methanol (1:2)- was the most effective. This provided data to point to the species of lipid involved.
Particularly, chloroform/methanol hi this ratio is used to extract neutral lipids, diacylglcerophospholϊpids and most sphingolipids from biological material with low lipid content.
Experiment 3
Enhancement of the Lipid Ring using Lead Salts Hairs were soaked in 0.1 mol/1 lead nitrate, at pH 5.8, at room temperature for 2 hours. Hairs were thoroughly washed in three successive 5 min immersions in MiIIiQ water, using the method of Bertrand et aϊ (Bertrand L, Doucet J3 Simionovici A, Tsoucaris G, Walter P3 2003. Lead revealed lipid organization in human hair BBA 1620: 218-224.). The results showed that the breast cancer ring was significantly enhanced in most cases. Figure 4 shows the diffraction pattern of a sample hair before and after lipid enhancement by lead nitrate. The ring after enhancement is much stronger than that before exposure to the lead nitrate.
The use of X-ray diffraction to establish the presence of an abnormality, in the form of a ring or halo, suggests that a patient from whom the sample is taken suffers from a condition which affects a keratin-containing component such as a hair. The present inventors have found that the associated ring in the pattern is due to a change in the lipid content of the keratin-containing component.
With this understanding, the inventors have found a method of enhancing an abnormal pattern to thereby provide a reliable diagnostic tool in the early detection of conditions including breast cancer.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
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 scope of the invention as broadly described.
The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.