WO2012155204A1

WO2012155204A1 - Assay for detection of telomerase rna

Info

Publication number: WO2012155204A1
Application number: PCT/AU2012/000543
Authority: WO
Inventors: Fabio Turatti; Minesh LALLA
Original assignee: Sienna Cancer Diagnostics Limited
Priority date: 2011-05-16
Filing date: 2012-05-16
Publication date: 2012-11-22

Abstract

A method for determining whether an individual has a disease, or is at risk of a disease, based on an assay to detect telomerase RNA (TR) isolated from TR bound in a telomerase enzyme complex.

Description

Assay for detection of telomerase RNA Field of the invention

The invention relates to telomerase and to the detection of cancer.

Background of the invention

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art. Telomerase activity is found in cancer cells, but not in non-cancer cells. Telomerase activity arises from the combined action of telomerase RNA (TR) with the catalytic protein telomerase reverse transcriptase (TERT) in the telomerase enzyme complex, resulting in primer extension of telomere ends.

Telomerase activity is found in cancer cells and not non-cancer cells, because TERT, the enzyme component that provides catalytic activity, is present in cancer cells only.

Other components of the telomerase enzyme complex, such as TR, are found in cancer cells and non-cancer cells in a form wherein they are not complexed with TERT (herein an 'unincorporated form'). This is why techniques that use exogenous reverse transcription (RT) to generate a detectable primer extension product (such as in TR RT- PCR) cannot discriminate between cancer cells and non-cancer cells.

Given expression of TERT in cancer cells only, the modernisation of TR RT-PCR has been to use endogenous TERT instead of exogenous TERT, as found in implementations including telomere repeat amplification p_rotocol (TRAP) and TBT (telomerase biosensor technology. Unlike TR RT-PCR, the extension product produced from TRAP and TBT is a function of TERT expression in cancer cells only, rather than a function of unincorporated hTR expression in cancer and ηόη-cancer cells. One limitation of techniques that use endogenous TERT is that TERT is generally found in very low copy number in each cell, so that very few copies of extension product are synthesised, meaning that detection of the product, and therefore detection of telomerase activity, becomes more difficult. For example, where extension product is to be detected by amplification (such as TRAP), fewer copies of extension product increase the likelihood that extension product will not be amplified, in which case false negatives become more likely, or otherwise signal to noise ratio is sub-optimal. The same issue applies to an improvement over TRAP, known as TBT whereby labelled nucleotide is incorporated into extension product The low copy number of functional TERT (i.e. TERT comprised in a telomerase enzyme complex) means that functional TERT cannot be detected directly; therefore, indirect detection via the detection of extension product, as in TR RT-PCR, TRAP and TBT is necessary.

At the time of the invention disclosed herein, one problem in the art was how to generate detectable amounts of cancer-specific extension product where exogenous RT (as in TR RT-PCR) cannot distinguish between cancer and non cancer cells, and endogenous RT in the form of TERT (as in TRAP and TBT) cannot generate detectable amounts of extension product.

Antibodies have been used to isolate the telomerase enzyme complex by binding to either the third component of the enzyme complex, dyskerin, or to reversibly-associated proteins such as TCAB and NAF1. These studies show that some antibodies (anti- NAF1 antibodies) do not isolate all TR (so that combined with TR RT-PCR, false negatives could occur), while other antibodies (such as anti-TCAB1) isolate both incorporated and unincorporated TR (so that combined with TR RT-PCT, false positives could occur).

Summary of the invention

In one embodiment there is provided a method for determining whether an individual has, or is at risk of disease, the method including the following steps: - providing a test sample from an individual in whom the presence or absence of disease, or risk of disease, is to be determined;

- contacting the test sample with an agent that binds to telomerase enzyme complexes and that does not bind to free TR; - separating the agent from the test sample so that a telomerase enzyme complex that has bound to the agent is separated from free TR in the test sample;

- providing conditions for releasing TR from a telomerase enzyme complex that has bound to the agent;

- modifying the released TR with an exogenous enzyme to improve detection of released TR;

- detecting for the presence of modified TR; wherein the presence of modified TR indicates that the individual has disease, or is at risk of disease.

In another embodiment there is provided a kit for use in the method described above, the kit including: an anti-telomerase enzyme complex antibody; an oligonucleotide (either RNA, DNA, or a synthetically modified form) for hybridising to TR; and written instructions for use in the method. Brief description of the figures

Figure 1 : ³²P-direct labelling assay indicating the efficiency of immuno-affinity purification. Figure 2: hTR Northern 'dot-blot of immuno-affinity purified material from HEK293T, GM847 and VA13 (10⁸ cell equivalents) cells. The top panel shows the dilutions of in vitro transcribed hTR XIV-62. The bottom panel shows the intensity of TR from HEK293T cells after immuno-affinity purification. Figure 3: Graph representing the relative intensity of the in vitro transcribed hTR standards. The HEK293T immuno-affinity purified material from 2 different sources are plotted and shown in red and green, respectively.

Figure 4: hTR detection using hTR primer pair in HEK293T and VA13 cell lines. The lanes are labelled according to the template used. Lane 1-11 : LMW marker (Genesearch); No template control; No RT control for hTR RNA from HEK293T IP material; hTR RNA from HEK293T IP material (102 bp); No RT control for hTR RNA from HEK293T total RNA; hTR RNA from HEK293T total RNA (102 bp); No RT control for RNA in VA13 IP material; RNA in VA13 IP material; No RT control for VA13 total RNA; VA13 total RNA and a 100 bp marker (Genesearch). VA13 cells are used as negative control as it is a telomerase-negative cell line.

Figure 5: ACTIN detection using ACTIN primer pair in HEK293T and VA13 cell lines. The lanes are labelled according to the template used. Lane 1-11 : LMW marker (Genesearch); No template control; No RT control for hTR RNA from HEK293T IP material; hTR RNA from HEK293T IP material; No RT control for hTR RNA from HEK293T total RNA; hTR RNA from HEK293T total RNA (294 bp); No RT control for RNA in VA13 IP material; RNA in VA13 IP material; No RT control for VA13 total RNA; VA13 total RNA (294 bp) and a 100 bp marker (Genesearch). VA13 cells are used as negative control as it is a telomerase negative cell line.

Figure 6: hTR and ACTIN detection using hTR and ACTIN primer pairs in HEK293T and VA13 cell lines. The lanes are labelled according to the template used. Lane 1-6 are reactions with hTR primers: LMW marker (Genesearch); No template control; hTR RNA from HEK293T IP material (102 bp); hTR RNA from HEK293T total RNA (102 bp); RNA in VA13 IP material; VA13 total RNA and Lane 7-11 are reactions with ACTIN primers: No template control; hTR RNA from HEK293T IP material; hTR RNA from HEK293T total RNA (294 bp); RNA in VA13 IP material; VA13 total RNA (294 bp) with lane 6 being left empty. VA13 cells are used as negative control as it is a telomerase negative cell line.

Figure 7: Chromatograph for sequencing data obtained from AGRF (5'-→3') using forward hTR primers for sample identified as HEK293T IP. The sequence depicted in the chromatogram has a 94% similarity to the hTR sequence available from NCBI (NR_001566.1).

Figure 8: Chromatograph for sequencing data obtained from AGRF (5'→3') using reverse hTR primers for sample identified as HEK293T IP. The sequence depicted in the chromatogram has a 98% similarity to the hTR sequence available from NBCI (NR_001566.1).

Detailed description of the embodiments

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. All of the patents and publications referred to herein are incorporated by reference in their entirety. In one embodiment there is provided a method for determining whether an individual has disease, or is at risk of disease. The method includes the following steps:

1. providing a test sample from an individual in whom the presence or absence of disease, or risk of disease, is to be determined; 2. contacting the test sample with an agent that binds to telomerase enzyme complexes and that does not bind to free TR;

3. separating the agent from the test sample so that a telomerase enzyme complex that has bound to the agent is separated from free TR in the test sample;

4. providing conditions for releasing TR from a telomerase enzyme complex that has bound to the agent;

5.. modifying the released TR with an exogenous enzyme to improve detection of released TR;

6. detecting for the presence of modified TR; wherein the presence of modified TR indicates that the individual has disease, or is at risk of disease.

It will be understood that the numbering applied above to each step is merely for ease of reference in the accompanying disclosure below. Further, as explained below, it will be understood that certain steps may be combined, substituted, or divided or drawn out into further steps. A. Definitions

Before turning to the relevant steps in detail, for purposes of interpreting this specification, the following definitions will generally apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall prevail.

"Telomerase enzyme complex" generally refers to a complex including telomerase reverse transcriptase (TERT) and telomerase RNA (TR). Dyskerin and other proteins such as TCAB and/or NAF1 may also be present in a telomerase enzyme complex. The telomerase complex adds TTAGGG repeats to the 3'-end of nucleic acid strands in telomere regions. Telomerase enzyme complexes are generally found in extremely low copy number in cancer cells (i.e. about 20-50 enzyme complexes per cell). "Telomerase¹' and "telomerase enzyme complex" are used interchangeably in the specification to mean the same thing.

"Telomerase reverse transcriptase" or "TERT" is the reverse transcriptase within a telomerase enzyme complex.

"Telomerase RNA" or "TR' is sometimes also referred to as "TERC. This is the RNA component of a telomerase enzyme complex from which telomerase primes to add repeats to telomere ends.

"hTR" refers to human TR.

"free TR or unbound TR" generally refers to TR that is not incorporated within a telomerase complex. As discussed herein, the inventors have found that free TR is widely expressed in cancer and non-cancer tissue. Therefore, free TR or unbound TR is not a biomarker of cancer.

"bound TR' generally refers to TR that is incorporated within a telomerase enzyme complex. Given that a telomerase enzyme complex is a biomarker of cancer, because it is found on cancer cells but not normal cells, bound TR, but not free TR or unbound TR, is a biomarker of cancer. "Modified TR" refers to a nucleic acid having a sequence that is at least substantially the same as the sequence of TR and/or fragments of TR sequences, or substantially complimentary to TR sequences and/or fragments of TR sequences. Modified TR may be a ribonucleotide and/or deoxyribonucleotide containing molecule.

"modifying the released TF? includes forming a partial or complete copy of the released TR or complimentary strand of the released TR in the form of a ribonucleotide and/or deoxyribonucleotide containing polynucleotides.

"exogenous enzyme" generally refers to enzyme that is not generated from the individual in whom the presence or absence of cancer or neoplastic disease is to be determined. One example of exogenous enzyme is enzyme that has been produced by recombinant means and/or commercially available. As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

B. Isolation of telomerase complexes from free TR.

Steps 2. and 3. of the above described method are critical insofar as the steps practically enable TR to be used as a biomarker of cancer. In more detail, as described herein, the inventors have found that free TR is expressed across a range of cancer and normal tissue so that free TR is not of itself a biomarker of cancer; i.e. a molecule that is expressed on cancer cells only, and not expressed on normal cells.

It is important that the product of step 3 is the removal of substantially all free TR from the sample. This is because if free TR remains with telomerase enzyme complexes when step 5. is performed, the exogenous enzyme could modify free TR so that a false positive result would be obtained. Therefore, the removal of substantially all free TR generally means the removal of enough free TR to prevent a false positive outcome. Put in other words, it is possible that some free TR may remain at the end of step 3., provided that the amount is not enough to give rise to a false positive result. An assay for detecting for the presence of free TR in a product of step 3 is described further herein. In a preferred embodiment herein, the inventors have used an antibody that has serological reactivity for a telomerase enzyme complex, specifically for TERT, as an agent that binds to telomerase enzyme complexes and that does not bind to free TR. Surprisingly, the inventors have found that an anti-TERT antibody enables the removal of substantially all free TR from bound TR.

Preferably the antibody is one having serological reactivity for TERT. One surprising finding according to the invention has been that anti-TERTantibodies do not spill or lose bound TR. This is surprising as earlier studies had suggested that anti-TCAB and anti- NAF1 antibodies do not capture all bound TR. The finding is important as loss of bound TR would increase the risk of a false negative result.

Antibodies having specificity for other telomerase complex components may be used.

Where an antibody is used as an agent that binds to telomerase enzyme complexes but not free TR, the antibody may be provided in any form, provided that it retains the complimentarity determining regions required for antigen specificity. Examples of suitable forms include dAb, Fab, Fd, Fv, F(ab')2, scFv, diabodies and CDR. The antibody may be a whole antibody or a portion of an antibody of any isotype. The antibody may be one obtained from monoclonal or polyclonal. The antibody may be produced by antisera, hybridoma, or by recombinant expression. The antibody may be chimeric, i.e. one containing human variable domains and non human constant domains. Alternatively, it may be humanized, i.e one formed by grafting non human CDRs onto a human antibody framework. Still further, the antibody may be fully human.

In another embodiment the agent that binds to telomerase enzyme complexes is a receptor based agent, a shark antibody, a macro or micro molecule, a fusion protein or ligand for receptor coupling. In one preferred form, an agent that binds to telomerase enzyme complexes but not free TR is attached to a solid phase so as to immobilise telomerase complexes thereon, thereby providing for separation of immobilised telomerase enzyme complexes from free TR, Examples of solid phases include any means having a surface upon which the agent may be based or adsorbed, such as a plate, dish, bead or the like.

It will be understood that it is not necessary to attach the agent to a solid phase if the result of binding of the agent to the telomerase complex is the formation of a substrate or precipitate that can be isolated from free TR. For example, a precipitate may be separated from free TR on the basis of differences in physical or chemical characteristics of TR and precipitate. Examples include separation on the basis of specific gravity, molecular size and/or charge.

In one embodiment, an agent for binding to telomerase enzyme complexes but not free TR is contacted with the test sample in the form that the sample exists in when obtained from the individual's body. For example, an antibody may be contacted directly with peripheral blood. Preferably the test sample is a sample that is derived from a biopsy of tissue or fluid. For example, if derived from urine, the test sample may be a re- suspension of relevant particles, in which case urine has been removed from the sample. Put in other words, a biopsy of tissue or fluid may be processed and the end product of that process may form a test sample.

One particularly useful processing step may be to enrich a test sample prior to step 1 for cancer cells by providing conditions enabling such enrichment. These conditions may include use of an agent, such as an antibody for binding cancer cells, examples of which include anti-EGF receptor (for tumour cells); anti-CD34 (stem cells); anti-CD45 (common leukocyte antigen); anti-CD 19 (pan-B-cell antigen) CD4 and CD8 (lymphocytes); anti-BerEP4 (pan-epithelial cell surface antigen); and anti-A33 (Colonic epithelial antigen). These antibodies may be provided on cell capture beads.

Otherwise the conditions may include the enrichment of any cells or cell fragments, particularly in circumstances where in a normal individual (i.e. individual not having cancer) one Would not expect to find cells in the relevant biopsy. Cells can also be purified or isolated by other methods such as continuous or non-continuous ficoll gradients for isolation of peripheral blood mononuclear cells (PBMC). In some embodiments, it will be necessary to deplete non-cancerous dividing cells from the test sample before further processing. These cells may have telomerase activity, and therefore bound TR. The types of cells of concern are dividing cells such as blasts and the like. Some of the above described antibodies can be used for this purpose. In circumstances where the individual is found not to have cancer, the antibody will not bind with telomerase enzyme complexes because there will essentially be no telomerase enzyme complex in the sample. Therefore, separation of the agent from the sample will remove the agent from the sample but not telomerase enzyme complexes, and where a further receptacle, vessel or the like is used to collect the agent, the receptacle will not contain telomerase enzyme complex either.

Importantly, the contact of the agent with the test sample is to be done in conditions that enable the binding of the agent to any telomerase enzyme complex that may be in the test sample. These conditions are generally known in the art. For example, where the agent is an antibody, it may be preferable to contact the antibody with the test sample at a temperature below room temperature and at physiological salt and pH for a time of about 1 to 2 hours.

With appropriate conditions, the outcome of step 3 is either no binding of the agent to telomerase enzyme complexes (because there are no telomerase enzyme complexes in the sample) or binding to telomerase enzyme complexes in the sample. The telomerase enzyme complexes may be separated from free TR in the test sample by removing TR from the test sample, in which case telomerase enzyme complexes, but not TR, remains in the test sample. Otherwise, telomerase enzyme complexes may be separated from free TR by removing telomerase enzyme complexes from the test sample, in which case the telomerase enzyme complexes are to be transferred from the test sample to a new receptacle or like for formation of a first reaction sample.

One alternative or additional step to steps 2 or 3 is the use of an agent or process step that enables either or all of the following:

(i) degrades free TR but not bound TR; (ii) binds to free TR but not bound TR, thereby permitting removal or separation of bound TR from free TR by removal of the agent;

(iii) separation based on a physical or chemical characteristic found in bound TR but not free TR or vice versa, examples of which include differential centrifugation, size exclusion chromatography, electrophoresis etc.

Therefore, in one embodiment the method may involve an additional step after steps 2 or 3 of utilizing an agent or process step for enabling one or more of outcomes (i) to (iii) above.

In other embodiments, the method may involve a step of utilizing an agent or process step for enabling one or more of outcomes (i) to (iii) above as ah alternative to steps 2 or 3.

Whether additional or alternative to steps 2 or 3, the telomerase enzyme complex (which may be bound or unbound to agent depending on which embodiment is applied) may be released in the same receptacle forming the test sample, or it may be transferred to the receptacle forming a 1st reaction sample.

In one embodiment, steps 1 to 3 may be combined to form a single step. For example, the test sample may be provided in a form whereby the biopsy is added to a receptacle or like that contains the agent and the first substantive step according to the method is therefore to separate the agent from the test sample. For example, a fluid biopsy could be added directly to a receptacle that contains an agent attached to a solid phase (for example an anti-telomerase complex antibody adsorbed to a bead) and an elution separation step then carried out.

In another embodiment, steps 2 and 3 may be combined into a single step, for example a chromatographic system whereby a portion of an earlier applied sample is eluted/separated as a latter applied portion is contacted with an agent.

C. Release of bound TR from telomerase complexes As discussed herein, steps 2 and 3 are critical insofar as they provide the basis on which bound TR, but not free TR, may be modified using an exogenous enzyme to become a detectable signal. The separation of bound TR from free TR means that the method does not have to rely on endogenous telomerase activity for signal creation, as in assays known in the prior art such as TRAP and like implementations. The reason why these latter assays rely on endogenous telomerase activity is because this activity is a cancer biomarker. However, the problem with these latter assays is that functional telomerase enzyme complexes (i.e. complexes capable of priming to form a telomeric repeat) are present in very low copy number in cancer cells. Therefore, for those assay methods that rely on endogenous telomerase, there is a risk that signal production will be low and therefore of false negative results, or at least a result that requires further clarification by further assay.

With steps 2 and 3, the invention provides the basis on which one can use an exogenous enzyme for signal generation, without the risk of generating a signal that is non-cancer specific. This risk is eliminated by steps 2 and 3 which remove non cancer specific TR (i.e. free TR) and retain cancer specific TR (i.e. bound TR), thereby ostensibly isolating a cancer biomarker in the form of bound TR. Thus, with the invention, the benefits that attain to the use of exogenous enzyme become possible.

A key benefit is that more copies of exogenous enzyme can be provided than the copy number of active telomerase enzyme complexes that normally exists in a cancer cell. This means that, unlike TRAP and like assays, according to the invention, the equilibrium of a modification reaction such as primer extension reaction can be forced toward obtaining maximum modification of all bound TR. The outcome is a stronger signal and therefore a higher likelihood of identification of true positives and a lower likelihood of occurrence of false negatives.

Another benefit is that a variety of detection formats in step 6 can then be selected, so that overall, the assay becomes much more flexible. For example, if the detection step has lower sensitivity, a greater amount of exogenous enzyme may be added. If the sensitivity is lower, a lesser amount of enzyme can be added. The end result is that the operator is able to select a range of detection steps each having different sensitivities including densitometry, luminescence, autoradiography etc.

Further, it becomes possible to use enzymes other than telomerase. For example, other reverse transcriptases could be used. It may even be possible to use enzymes that synthesise in either 5'— 3' or 3'— 5' directions.

None of the above benefits are attainable with the use of endogenous telomerase enzyme complexes.

As explained above, at the completion of step 3, the telomerase enzyme complex is in the test sample, i.e. it remains in the test sample; or it is transferred to a further sample from the test sample, such as a first reaction sample. In either case, it is substantially free from free TR. Therefore, at commencement of step 4, the relevant conditions for isolation or release of TR from telomerase complexes are applied to either the test sample or other sample such as a first reaction sample.

The objective of step 4 is to apply, or to provide conditions to the relevant sample that enable TR to be released from the telomerase enzyme complex should any complexes be in the relevant sample. Generally, it does not matter what conditions are provided as long as the TR is released in a substantially non degraded form - i.e. a form wherein it is capable of being modified by exogenous enzyme as in step 5 discussed below. Therefore, the following conditions may be applied: (i) those that selectively degrade or denature telomerase enzyme complex components other than TR;

(ii) those that compete TR from the telomerase complex.

It is preferred, although not essential, at the completion of step 4 that TR is isolated from telomerase enzyme complex components other than TR, i.e. TR remains but all other telomerase enzyme complex components are removed. This is because this may favour the activity of exogenous enzyme added in step 5, for example by ensuring that exogenous enzyme is free to couple with released TR, thereby maximising the likelihood of a modification of TR such as primer extension.

Where telomerase enzyme complex components remain after the TR has been released from the complex, the TR may remain in the receptacle in which it was when step 4 commenced (in which case telomerase complex components may have been either degraded in the receptacle or removed therefrom). Otherwise, the TR may be transferred from the receptacle to a further receptacle to form a second reaction sample in which case the telomerase complex components from which the TR has been released may be left in the test sample or further reaction sample or other. It will be understood that where the end result of step 3 is that there are no telomerase complexes in the test sample, 1st reaction sample or other, then the conditions that are applied to the relevant sample for release of TR from telomerase complexes will not result in TR being released into the relevant sample. This is because there is no telomerase enzyme complex in the relevant sample at the commencement of step 4. This applies in circumstances where the individual is found according to the method not to have neoplastic disease.

Isolating TR, the RNA component, from the telomerase enzyme complex is important in detecting the expression levels of TR. In one embodiment, the isolation of TR RNA is carried out using a column RNA extraction method that contains a built in DNase step to eliminate any genomic DNA contamination in the sample. The range of RNA extractions kits in the market are vast and most can be adapted. Variations to this isolation step might incorporate RNA extraction using a monophasic solution of phenol and guanidine isothiocyanate or even heat denaturation. Using heat might be used in conjunction with low salt concentration in order to dislodge the human telomerase RNA from the human telomerase enzyme complex and by doing this a one-tube protocol may be utilised in order to detect for TR by qRT-PCR. A one-tube reaction will revolve around the optimisation of the following steps: reverse transcription with a heat stable reverse transcriptase and the PCR reaction using molecular probes, FRET technology or fluorescent intercalating agents like SYBR green and Syto 9 to name a few. Another possibility would be a quantitative Northern blot assay by using a radioactively labelled RNA based probe and subsequently quantitated with a phosphorimager.

D. Enzymatic modification of released TR and detection of modified TR

The 5th step is important in circumstances where TR is found in the test sample or 2nd reaction sample (i.e. because telomerase enzyme complex was found in the test sample), because the TR will likely be present in quantities that cannot be detected without modification, this being the case because telomerase enzyme complexes containing the relevant TR are present in very low copy number in cancer cells. Therefore, exogenous enzyme is required to modify TR so that the modified TR is then detectable.

The TR may be modified by an enzymatic process that results in an improvement in the detection of the TR. The following modifications are examples:

(i) increasing the length of the TR,

(ii) incorporating or attachment of a label to the TR. In one embodiment, the modification is a form of primer extension. The primer extension may be in the 3' to 5' direction or the 5' to 3' direction. In one form, the extension does not involve a label. In this form, the objective is simply to create a template in the form of maximally extended TR upon which PCR amplification or probe hybridisation may be performed in step 6. In another form the extension may involve the use of labelled nucleotides or the like, in which case the following step 6 simply involves detection of label, or example by luminescence assay.

In one embodiment, the modification results in the attachment of a label to the TR. The reaction may involve phosphorylation or dephosphorylation. In this embodiment, the TR may not be extended at all. The label may be an intercalator such as ethidium bromide. In one embodiment, the exogenous enzyme is a polymerase such as those capable of extending TR in either 5' to 3' direction, or 3' to 5' direction. Preferably the polymerase is a polymerase with reverse transcriptase activity, examples of which include telomerase, terminal transferase and the like.

In another embodiment, the enzyme may be a kinase, phosphatase or other enzyme for attaching or removing secondary structure, such as methylation, to a nucleic acid. In one embodiment, a cDNA synthesis reaction is used consisting of a reverse transcriptase that operates between 30-60 °C and is deactivated at 70 °C. It is possible to obtain a reverse transcriptase that operates at much higher temperatures which will be used if ah RNA extraction step is replaced by a simple heat denaturation step. The primers for use in an assay may be gerie-specific but alternatives may include poly-dT primers. Random primers may also serve as an alternative. In the one-tube reaction the sample aliquot will be added to a reaction mix containing cDNA synthesis components as well as PCR components in order to amplify the target region of hTR. Variations to this step might be mended into a one-tube reaction using a heat stable reverse transcriptase or may involve using a commercial cDNA synthesis kit. Another variation after cDNA synthesis has taken place may include a radioactively labelled DNA probe that can be carried out in a Southern blot analysis and quantitated using a phosphorimager.

With appropriate conditions, the outcome of step 5 is either modification of TR (i.e. as in the case wherein at completion of step 4, TR has been released from telomerase complex) or no modification TR (because at the completion of step 5 there is no TR in the relevant sample).

Modified TR may be detected in step 6. by known techniques, examples of which are as follows: Step 5. modification of TR Step 6 detection assay

Primer extension of TR PCR amplification with label and detection by (without labelled nucleotide) luminescence

PCR amplification without label and detection by eye or densitometery

Hybridisation of labelled probe and detection by luminescence/autoradiography

Primer extension of TR Detection by luminescence/autoradiography

(with labelled nucleotide)

Phosphorylation of TR Detection by luminescence/autoradiography

Many other suitable detection formats are known in the art that could be used in step 6.

In one embodiment, the invention makes use of a detection system by amplifying the TR region and confirming size by agarose gel electrophoresis (with the use of molecular markers) and confirming the amplified region by sequencing. The variation to this current protocol will be optimised to have a qualitative output by using real-time PCR with molecular probes, FRET technology or fluorescent intercalating dyes. A one-tube reaction could well be another variation to the assay by using a qRT-PCR system. As mentioned above this assay will be more streamlined with a simple heat denaturation step as apposed to RNA extraction using columns and will contain both the components of cDNA synthesis as well as PCR components.

In an embodiment an antibody against modified TR could be used for detection. Detection methods may be based on any physical or chemical characteristic of modified TR such as by use of chromatography or spectroscopy.

In one embodiment, step 5 and step 6. may be combined into a single step. For example, TR may be modified with labelled nucleotide in an extension reaction that may be detected in real time i.e. modification of TR may be detected as the modification occurs. An example is real time PCR.

In one embodiment, steps 4 to 6 are combined by using a one step PCR process with a heat denaturation step prior to reverse transcription. The heat denaturation causes RNA extraction and the subsequent thermocycling causes reverse transcription and PCR all in one tube without addition of components further to commencement of the heat denaturation step. Examples of one step PCR include use of Tth DNA polymerase which can be used for reverse transcription and PCR, and the combination of AMV reverse transcriptase and Taq polymerase.

It will be understood that the outcome of step 6 is either the detection of modified TR (as applies where the relevant sample initially contained telomerase enzyme complexes containing bound TR) or no detection of modified TR (as applies where the relevant sample did not contain telomerase enzyme complexes containing bound TR, for example, as where the individual would be determined not to have neoplastic disease).

Thus, even where conditions are applied for modification of TR and detection thereof, the outcome may be no modification of TR (because there is no TR to modify in the sample to which the conditions are applied in step 5) and therefore no detection of modified TR.

In another embodiment, the outcome of step 6 may be no detection at levels below a threshold observed in normal cells. In one embodiment, steps 5 and 6 are as follows:

5. modifying the released TR with an exogenous enzyme, thereby forming a detectable product; 6. detecting for the presence of the detectable product from step 5. E. Detecting & monitoring cancer

As described herein, the method of the invention is particularly useful for determining whether an individual has neoplastic disease, or in other words, for detecting or diagnosing disease.

The method also finds application in prognosis, evaluation and monitoring of disease treatment.

The method may find application regarding any disease where telomerase enzyme complex expression is a disease biomarker. Generally these diseases are neoplastic diseases, i.e diseases involving the unregulated proliferation of cells and/or the formation of cells having abnormal structure and/or function. Examples include pre- neoplasia, early cancer, non-invasive cancer, carcinoma in situ, premalignancy, invasive cancer, advanced cancer and metastatic cancer.

Broad examples include breast tumors, colorectal tumors, adenocarcinomas, mesothelioma, bladder tumors, prostate tumors, germ cell tumor, hepatoma/cholongio, carcinoma, neuroendocrine tumors, pituitary neoplasm, small round cell tumor, squamous cell cancer, melanoma, atypical fibroxanthoma, seminomas, nonseminomas, stromal leydig cell tumors, Sertoli cell tumors, skin tumors, kidney tumors, testicular tumors, brain tumors, ovarian tumors, stomach tumors, oral tumors, bladder tumors, bone tumors, cervical tumors, esophageal tumors, laryngeal tumors, liver tumors, lung tumors, vaginal tumors and Wilm's tumor.

Examples of particular cancers include but are not limited to adenocarcinoma, adenoma, adenofibroma, adenolymphoma, adontoma, AIDS related, cancers, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenocystic carcinoma, adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft-part sarcoma, ameloblastoma, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, angioma sclerosing, angiomatosis, apudoma, anal cancer, angiosarcoma, aplastic anaemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer, brain stem glioma, brain and CNS tumors, breast cancer, branchioma, CNS tumors, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancers, cutaneous T-cell lymphoma, carcinoma (e.g. Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, Merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell), carcinosarcoma, cervical dysplasia, cystosarcoma phyllodies, cementoma, chordoma, choristoma, chondrosarcoma, chondroblastoma, craniopharyngioma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, dermatofibrosarcoma- protuberans, desmoplastic-small-round-cell-tumor, ductal carcinoma, dysgerminoam, endocrine cancers, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extra-hepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, fanconi anaemia, fibroma, fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinal cancers, gastrointestinal-carcinoid-tumor, genitourinary cancers, germ cell tumors, gestationaltrophoblastic-disease, glioma, gynaecological cancers, giant cell tumors, ganglioneuroma, glioma, glomangioma, granulosa cell tumor, gynandroblastoma, haematological malignancies, hairy cell leukemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharynx cancer, hamartoma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, hemangiosarcoma, histiocytic disorders, histiocytosis malignant, histiocytoma, hepatoma, hidradenoma, hondrosarcoma, immunoproliferative small, opoma, ontraocular melanoma, islet cell cancer, Kaposi's sarcoma, kidney cancer, langerhan's cell-histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, li-fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leigomyosarcoma, leukemia (e.g. b-cell, mixed cell, null-cell, t-cell, t-cell chronic, htlv-ii-associated, lymphangiosarcoma, lymphocytic acute, lymphocytic chronic, mast-cell and myeloid), leukosarcoma, leydig cell tumor, liposarcoma, leiomyoma, leiomyosarcoma, lymphangioma, lymphangiocytoma, lymphagioma, lymphagiomyoma, lymphangiosarcoma, male breast cancer, malignant- rhabdoid-tumor-of-kidney, medulloblastoma, melanoma, Merkel cell cancer, mesothelioma, metastatic cancer, mouth cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplasia syndromes, myeloma, myeloproliferative disorders, malignant carcinoid syndrome carcinoid heart disease, medulloblastoma, meningioma, melanoma, mesenchymoma, mesonephroma, mesothelioma, myoblastoma, myoma, myosarcoma, myxoma, myxosarcoma, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen breakage syndrome, non-melanoma skin cancer, non-small-cell-lung-cancer-(nsclc), neurilemmoma, neuroblastoma, neuroepithelioma, neurofibromatosis, neurofibroma, neuroma, neoplasms (e.g. bone, breast, digestive system, colorectal, liver), ocular cancers, oesophageal cancer, oral cavity cancer, oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasal cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral- neuroectodermal-tumors, pituitary cancer, polycythemia vera, prostate cancer, osteoma, osteosarcoma, ovarian carcinoma, papilloma, paraganglioma, paraganglioma nonchromaffin, pinealoma, plasmacytoma, protooncogene, rare-cancers-and-associated- disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, reticuloendotheliosis, rhabdomyoma, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (sclc), small intestine cancer, soft tissue sarcoma, spinal cord tumors, squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma, sarcoma (e.g. Ewing's experimental, Kaposi's and mast-cell sarcomas), Sertoli cell tumor, synovioma, testicular cancer, thymus cancer, thyroid cancer, transitional-cell-cancer-(bladder), transitional-cell-cancer-(renal-pelvis-/-ureter), trophoblastic cancer, teratoma, theca cell tumor, thymoma, trophoblastic tumor, urethral cancer, urinary system cancer, uroplakins, uterine sarcoma, uterus cancer, vaginal cancer, vulva cancer, Waldenstrom' s-macroglobulinemia and Wilms' tumor.

The method may be applied to a range of biological samples, examples of which include urine and sedimented cells in urine, bladder washings, renal pelvic washings, exfoliated faecal epithelial cells, endoscopic biopsy specimens, bone marrow, peripheral blood, fine needle aspirates, bronchial alveolar lavage, bronchial brushings and washings, sputum, scrapings and smears, biopsies and tissue sections, nipple discharge, cerebrospinal fluid and peritoneal washings. In more detail, typical clinical samples that may be analysed according to the method of the invention include, but are not restricted to, the following:

• bronchial alveolar lavage, bronchial brushings and washings, sputum, scrapings, smears for the detection of neoplasms in the bronchial tree, lung cancer, head and neck cancer.

• fine needle aspirates, biopsies and tissue sections for the detection of malignant cells in the lung, lymph nodes, pancreas, salivary gland, breast, liver, thyroid, and in prostate cancer.

• sedimented cells in urine, renal pelvic washings, bladder washings for the detection of prostate cancer, bladder cancer, urogenital tract cancer, and renal cancer.

• blood for the detection of melanoma and cancers of the haematopoietic system.

• body cavity fluids (pleural fluid, peritoneal fluid, pericardial fluid, peritoneal washings, gutter washings) for the detection of malignant neoplasms. · cerebrospinal fluid for the detection of malignant cells in the CSF.

• endoscopic biopsy specimens for the detection of cancer of the gastrointestinal tract.

• faecal specimens for the detection of malignant cells in colon cancer and other cancers of the gastrointestinal tract. · nipple Discharge: for the detection of breast cancer and cancers causing nipple discharge.

• PAP Test™ / PAP smears (Cervical/Vaginal Screening) for the detection of cervical, vaginal and ovarian cancer. May also be used for the detection of certain infectious and inflammatory conditions. • skin (TZanck Smear) for vesicular diseases secondary to herpes virus infections (Herpes Simplex virus and Varicella-Zoster virus).

In the case of bladder cancer, tumor epithelial cells are isolated by selective capture from urine using epithelial cell-specific antibodies attached to magnetic beads. The method is particularly applicable to those forms of neoplastic disease whereby a sample in the form of a fluid can be obtained without requirement for biopsy or other clinical or surgical intervention.

The individual is typically a mammal, preferably human, or a companion animal such as a cat or dog. Examples

Example 1 Detection of incorporated (bound) and unincorporated (free) hTR RNA in HEK293T and VA13 cells.

Materials and Methods:

Cell Culture Two human cancer cell lines, accepted as positive and negative cell lines for human telomerase were used in this study. The embryonic cancer cell line, HEK293T (ATCC) and a telomerase negative cell line, VA13 (ATCC) were grown and harvested using 0.25% Trypsin (GIBCO). Cell pellets were then washed with phosphate buffered saline (1xPBS, GIBCO) before being stored at - 80°C. Immuno-affinity purification of human telomerase

Immuno-affinity purification of human telomerase was carried out on cell pellets (10⁸ cells) of HEK293T and VA 3 cell lines and telomerase was quantified by hTR Northern 'dot blot'. Cell pellets of 10^B cells were resuspended in 5 mL (20,000,000 cells/mL) standard telomerase buffer: HEPES-KOH (pH 8.0) = 20 mM; KCI = 300 mM; MgCI₂ = 2 mM; Triton X-100 = 0.1% v/v; Glycerol = 10% v/v; PMSF = 1 mM; DTT = 1 mM.

Suspensions were rotated at 4°C for 30 minutes after which the lysates were clarified with centrifugation at 3,000 rpm at 2°C for 20 minutes. Each lysate (5 mL) was treated with 100 pg of anti-hTERT antibody (20 pg/mL final concentration, XIV-60a). The solutions were rotated at 4°C for 30 minutes. One hundred pL Roche Protein G beads (200 pL of 50% slurry) was added to each sample and rotated at 4°C for 1 hour. Each lysate/Protein G suspension was then collected into two micro-spin columns (50 pL beads per column). Each column was then washed with 10 mL cold buffer with vacuum suction, followed by a brief spin to remove traces of buffer. The bead samples were suspended in 200 pL telomerase buffer (minus PMSF) containing antigenic peptide (5 pL, 1mM in 0.02% NaN₃ = 5 nmoles). The suspensions were rotated at room temperature for 1 hour. Immuno-affinity purified products were collected with brief centrifugation. hTR Northern 'dot-blot'

A quantitation standard of in vitro transcribed hTR XIV-62 was used and each sample was made up with 10 pL TE + 90 pL Formamide/TBE buffer with the following dilutions: 0.2, 0.4, 0.6, 0.8, LO fmole.

Ten pL (2.5 x 10⁶ cell equivalents) of IP sample was diluted into 90 pL Formamide TBE buffer for hTR blotting. Immuno-affinity purified HEK293T telomerase was also blotted with a telomerase solution at a concentration of 1.5 * 10^s cell equivalents/pL This dilution was then diluted three-fold with buffer to 0.5 χ 10⁶ cell equivalents/pL. A 5 pL aliquot (2.5 χ 10⁶ cell equivalents) and 5 pL buffer diluted into 90 pL Formamide TBE buffer. All samples were heated at 70°C for 10 minutes and immediately placed onto ice. The samples were then blotted onto Amersham Hybond N+ and air-dried for ~1 hour and "auto-crosslinked" with UV (254 nm) three times. The samples were then hybridized in 25 mL Church buffer at 55 °C over weekend with ~10⁷ cpm probe: 5'- ³²PCGG TGG AAG GCG GCA GGC CGA GGC-3' (ΧΝΛ31) and washed 3 times with 50 mL per wash in (0.1 xSSC + 0.1% w/v SDS) for 10 minutes per wash at room temperature.

RNA isolation

(i) RNA extraction of immuno-affinity purified telomerase Cell pellets containing 10⁸ cells from HEK293T and VA13 cell lines were immuno-affinity purified. Each of the cell pellets were eluted to a final volume of 400 pL.

VA13 is a negative cell line for telomerase whilst the HEK293T yielded ~0.4 fmoles of telomerase per 10 μΙ_.

Sixty μΙ_ of HEK293T IP and 100 μΙ_ of VA13 IP were used in RNA extraction steps. Therefore 2.4 fmoles of telomerase (HEK293T) was added to the RNA extraction step.

1. IP material (HEK293T and VA13) was allowed to thaw and immediately placed on ice.

2. Manufacturer's instructions were followed when isolating RNA from IP material. RNA extraction kits from Macherey-Nagel (Nucleospin RNA XS) were used. 3. RNA was isolated using the Nucleospin RNA XS kit yielded the following volumes: a. HEK293T IP material was eluted to a final volume of 30 μΙ_. b. VA13 IP material was eluted to a final volume of 30 μΐ_.

(ii) RNA extraction of total RNA from cultured cell pellets

Cell pellets from HEK293T (10⁶) and VA13 (5 x 10⁷) cells were used in the total RNA extraction step to act as controls for the assay whereby they were tested for the housekeeping gene, actin. 1. HEK293T and VA13 cell pellets were allowed to thaw and immediately placed on ice.

2. Manufacturer's instructions were followed when isolating total RNA from cultured cells. RNA extraction kit from Macherey-Nagel (Total RNA Isolation Nucleospin RNA II) was used.

3. RNA isolated using the Nucleospin RNA II kit yielded the following volumes: a. 10⁶ HEK293T cells were eluted to a final volume of 60 μΐ_. b. 5 x 10⁷ VA13 cells were eluted to a final volume of 480 μΙ_. (iii) cDNA synthesis (Reverse Transcription-RT reaction)

1. The following components were added into a sterile 0.5 ml. tube: a. 50 μΜ (1 pL) hTR R 26 (for IP material) and random primers (for total RNA). i. I P HEK293T and VA13 were set up with hTR R 26. ii. VA13 and HEK293T (total RNA) were set up with random primers. b. 5 μΐ_ RNA (concentration undetermined) c. 1 pL (10 mM) dNTP mix. d. 6 μΙ_ dh^O to make a final volume of 13 μΙ_.

2. Samples were heated for 5 minutes at 65 °C and placed on ice for 1 minute.

3. The contents were collected and gently centrifuged.

4. The following components were added to the mix: a. 4 μΙ_ 5x First Strand Buffer. b. 1 μΙ_ 0.1Μ DTT. c. 1 μΙ RNaseOUT Recombinant RNase Inhibitor d. 1 iL Superscript III RT

5. The samples were mixed by pipetting up and down and incubated at 53°C for 60 minutes.

6. The samples were inactivated by incubating at 70°C for 15 minutes.

7. A minus RT control reaction was set up for each sample. cDNA samples were diluted 1 in 5 and made up to a final volume of 100 pL.

8. The reactions were stored at -80°C until PCR was set up. The list below indicates the identification of each sample:

SCD-PCR-048 HEK IP HTR 26 NO 100 uL

RT

SCD-PCR-049 HEK IP HTR 26 RT 100 uL

SCD-PCR-050 V IP HTR 26 NO RT 100 uL

SCD-PCR-051 V IP HTR 26 RT 100 uL

SCD-PCR-052 HEK TOTAL RP NO 100 uL

RT

SCD-PCR-053 HEK TOTAL RP RT 100 uL

SCD-PCR-054 V TOTAL RP NO 100 uL

RT

SCD-PCR-055 V TOTAL RP RT 100 uL (iv) Polymerase chain reaction (PCR) for hTR and Actin PCR components: a) hTR F 20 (10 μΜ) - Sigma Aldrich - (G1704-070) b) hTR R 26 (10 μΜ) - Sigma Aldrich - (G1704-068) c) ACTIN F 294 - Sigma Aldrich - (G2161 -074) d) ACTIN R 294- Sigma Aldrich - (G2161-075) e) dNTPs (10 mM) - Invitrogen - Lot* 397985 f) iProof High Fidelity DNA Polymerase (20U) - BIORAD - Lot# 530034

PCR was carried out on both the RNA obtained from IP material of HEK293T and VA13 cell lines.

The component of RNA that was obtained from HEK293T cells is known as the incorporated hTR component of the human telomerase complex. VA13 IP material does not contain the hTR component and is used as a viable control as VA13 is a telomerase negative (hTERT -ve and hTR- ve) cell line. The RNA obtained from cell lysates of HEK293T and VA13 are total RNA and contains the unincorporated hTR component due to the human telomerase complex being lost during the RNA extraction. This means incorporated hTR is lost.

The cDNA prepared from these RNA samples (hTR RNA and total RNA) were subject to PCR using primers designed around the hTR and actin genes. Primers used for hTR detection: Forward 5 -AGG CGC CGT GCT TTT GCT CC-3' and reverse 5'-GTT TGC TCT AGA ATG AAC GGT GGA AG-3", yielding a product of 102 bp and ACTIN: Forward 5 -GGA CTT CGA GCA AGA TAT GG-3' and reverse 5'- GCA GTG ATC TCC TTC TGC ATC-3,' yielding a product size of 294 bp. PCR reactions contained 1x iProof HF buffer, 0.5 mM dNTPs, 0.5 μΜ primers and 0.02 U/μΙ- iProof DNA polymerase in a final reaction volume of 20 μΙ_. PCR cycling conditions were optimised, on a BIORAD MJ Mini Thermal Cycler, for both sets of primers using denaturation at 98°C for 30 seconds, followed by 35 cycles of denaturing at 98°C for 10 seconds, annealing at 65°C for 20 seconds, extension at 72°C for 20 seconds which was then followed by a final extension step of 72°C for 10 minutes. PCR products were then subjected to gel electrophoresis using a 2% agarose gel and analysed using a UV filter on the BIORAD ChemiDoc Imaging system.

Results (i) Immuno-affinity purification of telomerase from cultured cells

Telomerase was immuno-affinity purified from 10⁸ HEK293T cells and was subjected to ³²P-direct labelling assay to test for telomerase activity and hence the efficiency of immuno-affinity purification. Figure 1 shows the efficiency of immunoaffinity purification as the activity is comparable to an immuno-affinity purified positive control. (ii) hTR Northern 'dot-blot'

A standard of transcribed hTR XI -62 was compared to the immuno-affinity purified material from G 847, (another telomerase negative cell line), VA13 and HEK293T cells. In Figure 2 the in vitro hTR standards demonstrate good linearity. HEK293T immuno-affinity showed a lower intensity then the HEK293T immuno-affinity purified at another source. The latter has -0.4 fmoles hTR per 2.5 χ 10⁶ cell equivalents; the HEK293T telomerase prepared at the former source is lower, -0.05 fmoles per 2.5 * 10⁶ cell equivalents. The GM847 and VA13 samples are negative, as expected. Figure 3 demonstrates the linearity of the transcribed hTR standards and the immunopurification of both HEK293T cell lines at both sources. (iii) Detection of the presence of hTR RNA and actin in both IP material and total RNA in HEK29T and VA13 cells cDNA was made in a 20 pL final volume. This was then diluted five-fold and made up to a final volume of 100 pL with dh^O. Two μΙ was used in a 20 pL PCR reaction. Controls without template were used in each set up to evaluate primer dimer formations for each primer set and to act as a contamination control. Reactions containing samples are also shown in the figures. In Figure 4 and 5 a "minus RT" (reverse transcriptase) control is used to assess the cDNA synthesis reaction for each IP and total RNA reaction. Figure 4 and 5 are the reactions using the hTR and actin primers respectively. In Figure 6 the same experimental protocol was used together with the same cDNA samples and the experiment was repeated without the "minus RT" controls. (iv) Sequencing the PCR products to confirm hTR

PCR products together with forward and reverse primers were submitted to AGRF (Australian Genome Research Facility) to confirm the detection of hTR amplification. The samples shown in Figure 4 and denoted HEKIP and HEKIP (No RT) were submitted to the AGRF. Sequencing data was then cross referenced using the National Centre for Biotechnology Information ((NCBI) database to confirm the sequence data obtained from AGRF. Below is the data obtained from AGRF and the confirmation of the output data when cross referenced from NCBI. Sequences were also entered into Chromatogram Explorer version 3.1.1 in order to observe the data obtained from AGRF.

Figure 7 and 8 are a representation of the data obtained for the forward and reverse directions both of which show >94% similarity to the hTR sequence available with NCBI (NR_001566.1).

(v) Sequence obtained from AGRF (5'-3') with sequencing in the forward direction (Figure 7)

5'CCA GCG ATG CTT TGC TCC GCG CGC CGC CTT CCA CCG TTC ATT CTA GAG CAA CAA AAA ATG TCA GCT GCT GGC ACC AAA CCT TTA GAT-3'

Cross referenced with the following sequence: NR_001566.1 - Homo sapiens telomerase RNA component (TERC), non-coding RNA (Lehgth=451) Output readout

^• GENE ID: 7012 TERC | telomerase RNA component [Homo sapiens]

^• Score = 86.0 bits (94), Expect = 4x1ο^6"15 Identities = 55/59 (94%), Gaps = 1/59 (1%)Strand=Plus/Plus (vi) Sequence obtained from AGRF (5'-3') with sequencing in the reverse direction (Figure 8)

5'-TTT TAG CTG ATG ACG GTG GAG GCG GCG CGC GGG GAG CAA AGC ACG GCG CCT ACG CCC TTC TCA GTT AGG GTT AAA ACA TCC ATA AAT GGC G-3'

Cross referenced with the following sequence: NR_001566.1 - Homo sapiens telomerase RNA component (TERC), non-coding RNA (Length=451)

Output readout

^• GENE ID: 7012 TERC | telomerase RNA component [Homo sapiens]

^• Score = 82.4 bits (90), Expect = 5x1 O*^"14 Identities = 49/50 (98%), Gaps = 1/50 (2%)Strand=Plus/ inus Example 2 Various sample preparation steps

Urine processing procedure

All steps are performed on ice to prevent the non-specific attachment of cells to the Dynal beads.

1. Urine is collected and kept on ice. The urine is transferred to a 50 ml_ tube. If there is more than 50 ml_, the urine is divided into 2 equal volumes in the 50-mL tubes and each processed as below. 2. Sample is centrifuged at 750 g for 5 minutes at 4°C. Supernatant is discarded.

3. Pellet is resuspended in 10 mL PBS (pH 7.4), supplemented with 0.1% w/v BSA and a protease inhibitor tablet (thereafter referred to as wash buffer).

4. Sample is centrifuged at 750 g for 5 minutes at 4°C. Supernatant is discarded into the beaker with the HazTab.

5. Washing step is repeated (steps 4-5).

6. During step 5, the Epithelial Enrich Cellection Dynal beads are washed once with 100μΙ wash buffer (using the Dynal magnetic trap).

7. Following centrifugation, the pellet is re-suspended in 1 mL of wash buffer and transferred to a 1.5-mL eppendorf tube.

8. Washed beads are added to the washed urine cells from Step 5. For pellets that are less than 1 mm in diameter, 25 pi of beads are used. For pellets between 1-2 mm, 30 μΙ of beads are used. For anything larger than 2 mm, 40 μΙ of beads are used.

9. The beads and urine cells are mixed gently for 30 minutes at 4°C with rotation (60 r.p.m.).

10. Samples are centrifuged (Capsule Tomy HF120) for 30 sec to ensure that no beads or buffer is left in the lid of the Eppendorf tube.

11. Tubes are placed in the Dynal Magnetic Trap (Dynal MPC-S), and the supernatant carefully transferred to a fresh 1.5- mL Eppendorf tube using a Gilson P1000 pipette: Supernatant is centrifuged at 13,000 r.p.m. in a Hereaus Biofuge for 5 minutes at 4°C. Supernatant is removed and cells in the pellet lysed (this contains cells that have not bound to the Epithelial Enrich Cellection Dynal beads). This fraction may contain activated lymphocytes and should be stored separately as a frozen cell pellet (-70°C) for subsequent analysis, if required. 12. Beads from Step 11 are washed by re-suspending in I mL wash buffer and then the supernatant is removed using the Dynal magnetic trap as described above. This supernatant is discarded. 3. CHAPS lysis buffer (I00 μΙ) is added to the Dynal beads bound to the epithelial cancer cells.

14. Cells are lysed by pipetting up and down at least 10 times using a Gilson P200 pipette.

15. Lysates are incubated on ice for 30 minutes.

16. Lysates are centrifuged at 13,000 r.p.m. in a Hereaus Biofuge for 5 minutes at 4°C. 17. Beads are removed by place tubes in the magnetic trap.

18. Supematants (~30 μΙ) are aliquotted into each of 3 tubes and the pellet discarded.

19. Lysates are snap-frozen on dry ice for 5 minutes and transferred to -70°C refrigerator.

Faeces processing procedure

Faecal samples are collected under informed consent from patents with clinically proven colorectal cancer. Samples are collected at home and transported immediately to the laboratory (less than 2 hours) where aliquots (2 g) are dispersed in Puck's saline with antibiotics (500 LVL penicillin, 500 mg/L Streptomycin-sulphate, 1.25 mg/L amphotericin B and 50 mg/L gentamicin). The faecal slurry is filtered sequentially through 100-pm and 60- pm membranes (Nylon/Net membrane filters, Millipore, Australia) to remove large debris before being centrifuged at 400 g for 10 minutes at 4°C. The pellet is washed twice with PBS containing 1% v/v FCS and 0.6% w/v sodium citrate, followed by recovery of epithelial cells using 40 μΙ Epithelial Enrich CELLection Dynabeads. The cells are incubated with the Dynabeads for 30 min at 4°C and the supernatant then removed using the Dynal Magnetic Particle Processor. The cells attached to the magnetic beads are washed 3 times with PBS containing 0.1% w/v BSA before lysis with 200 μΙ CHAPS lysis buffer. The resulting supernatant is snap frozen in liquid nitrogen and stored at -70°C.

Leukemia cells processing procedure

Cells for diagnosis and analysis of leukemia patients are isolated from bone marrow or peripheral blood. Ten-mL human bone marrow aspirates, taken from the iliac crest of normal donors, are diluted 1 :1 with phosphate-buffered saline and centrifuged at 900 g for 10 minutes at room temperature. The washed cells are resuspended in PBS to a final volume of 10 mL and layered over an equal volume of 1.073 g/mL Percoll solution. After centrifugation at 900 g for 30 minutes, the mononuclear cells (MNCs) are recovered from the gradient interface and washed with PBS. Percoll-fractionated MNCs or non-fractionated bone marrow cells are suspended in PBS for analysis. MNCs are isolated from buffy coats of peripheral blood by Ficoll-Paque density gradient centrifugation and washed in PBS.

Claims

The claims defining the invention are as follows:

1. A method for determining whether an individual has disease, or is at risk of disease, the method including the following steps:

- providing a test sample from an individual in whom the presence or absence of disease, or risk of disease is to be determined;

- contacting the test sample with an agent that binds to telomerase enzyme complexes and that does not bind to free TR;

- separating the agent from the test sample so that a telomerase enzyme complex that has bound to the agent is separated from free TR in the test sample; - providing conditions for releasing TR from a telomerase enzyme complex that has bound to the agent;

- providing conditions for modifying the isolated TR with an exogenous enzyme to improve detection of released TR;

2. The method according to claim 1 wherein the agent is an antibody.

3. The method according to claim 2 wherein the antibody has serological reactivity with a telomerase enzyme complex.

4. The method of claim 3 wherein the antibody has serological reactivity with TERT.

5. The method of any one of the preceding claims wherein the separation of the agent from the test sample results in the removal of substantially all free TR from TR bound within telomerase enzyme complexes.

6. The method of any one of the preceding claims wherein the agent that binds to the telomerase enzyme complex is attached to a solid phase.

7. The method of any one of the preceding claims including a step of enriching the test sample for cells, preferably for neoplastic cells.

8. The method of any one of the preceding claims including an additional step of:

- degrading free TR but not bound TR; - utilizing an agent for binding to free TR but not bound TR and removing said agent from the test sample; or

- separating free TR from bound TR on basis of a physical or chemical characteristic found in free TR but not bound TR.

9. The method of any one of the preceding claims including utilizing an agent for binding to free TR but not bound TR and removing said agent from the test sample as an alternative to the steps of:

- separating the agent from the test sample so that a telomerase enzyme complex that has bound to the agent is separated from free TR in the test sample.

10. The method of any one of the preceding claims wherein steps 1 to 3 are combined into a single step.

11. The method of any one of the preceding claims wherein steps 1 and 2 are combined into a single step.

12. The method of any one of the preceding claims wherein steps 4 to 6 are combined into a single step.

13. The method of any one of the preceding claims where TR is released from telomerase enzyme complex by either or both of:

- selectively degrading or denaturing telomerase enzyme complex components other than TR;

- utilizing an agent to compete TR from the telomerase enzyme complex.

14. The method of any one of the preceding claims wherein all telomerase enzyme complex components other than TR are depleted from the sample after release of TR from a telomerase enzyme complex.

15. The method of any one of the preceding claims wherein TR is modified by either or both of: - increasing the length of TR;

- incorporating or attaching a label to TR.

16. The method of any one of the preceding claims wherein TR is modified by a primer extension reaction.

17. The method of claim 16 wherein the primer extension reaction extends in the 3' to 5' direction.

18. The method of any one of the preceding claims wherein the exogenous enzyme is an RNA dependent polymerase.

19. The method of any one of the preceding claims wherein real time PCR is used to form cDNA from TR.

20. The method of any one of the preceding claims wherein the disease is neoplasia.

21. The method of claim 20 wherein the neoplasia is cancer.

22. The method of claim 21 wherein the cancer is bladder cancer.

23. The method of claim 1 wherein as an alternative to the step of contacting the test sample with an agent that binds to telomerase enzyme complexes and that does not bind to free TR, the method includes the step of contacting the test sample with an agent that degrades free TR but not bound TR.