NZ620854B2 - Prognostic methodology - Google Patents

Abstract

prognostic method for determining the progression of a disease including or characterised by telomere shortening is disclosed. The method comprises i) using high-resolution telomere length analysis to determine the longest mean telomere length at which telomere end-end fusion events can be detected in samples of tissue from a number of individuals presenting with the same disease, in order to identify a threshold figure that represents an indication of the mean telomere length at which telomeres become dysfunctional and capable of fusion; ii) determining the prognostic mean telomere length of samples of tissue from a number of individuals presenting with the disease, by taking those samples whose mean telomere length is less than the threshold and averaging the mean telomere length of those samples; iii) determining the mean test telomere length of a sample taken from a patient suspected of having or presenting with the disease and, where the mean test telomere length is less than the prognostic mean telomere length, concluding time to first treatment is poor and/or response to treatment is poor and/or overall survival is poor; or iv) determining the mean test telomere length of a sample taken from a patient suspected of having or presenting with the disease and, where the mean test telomere length is greater than the prognostic mean telomere length, concluding time to first treatment is good and/or response to treatment is good and/or overall survival is good ed in samples of tissue from a number of individuals presenting with the same disease, in order to identify a threshold figure that represents an indication of the mean telomere length at which telomeres become dysfunctional and capable of fusion; ii) determining the prognostic mean telomere length of samples of tissue from a number of individuals presenting with the disease, by taking those samples whose mean telomere length is less than the threshold and averaging the mean telomere length of those samples; iii) determining the mean test telomere length of a sample taken from a patient suspected of having or presenting with the disease and, where the mean test telomere length is less than the prognostic mean telomere length, concluding time to first treatment is poor and/or response to treatment is poor and/or overall survival is poor; or iv) determining the mean test telomere length of a sample taken from a patient suspected of having or presenting with the disease and, where the mean test telomere length is greater than the prognostic mean telomere length, concluding time to first treatment is good and/or response to treatment is good and/or overall survival is good

Description

Prognostic Methodology The invention relates to a novel prognostic method for determining at least one, or a combination, of the following: time to first treatment, response to treatment or overall survival for a patient ting with a disease including or characterised by telomere shortening, comprising an assessment of the longest mean telomere length at which telomere end-end fusion events can be detected and then a determination of the mean telomere length in the fusogenic range (i.e. the range below said mean telomere length at which telomere end-end fusion events can be detected) and the subsequent use of the mean telomere length in the fusogenic range as a prognostic indicator. The invention also relates to the use of said method in a treatment regimen.

Background of Invention Chronic cytic mia (CLL) is the most common adult leukaemia, characterised by the lation of immuno-incompetent, monoclonal CD5+ B- lymphocytes. CLL has a very heterogeneous clinical course with survival ranging from a few months to many decades. Treatment strategies vary with g and e progression and include chemotherapy, radiotherapy, monoclonal antibody therapy or bone marrow transplantation, with early stage patients often receiving no treatment. Early al ention is required for patients with an sive form of the disease, whereas patients with more benign forms simply need monitoring for disease progression at which point appropriate treatment may be administered. In this latter respect, it has been shown that early stage CLL ention does not improve survival rates. It is therefore inappropriate to expose someone presenting with a e that is unlikely to be life-threatening for up to 30 years with highly dangerous chemotherapeutic drugs. A reliable method for distinguishing the various forms of the disease is therefore desirable. Although the Binet and Rai staging s are le predictors of clinical outcome between the staging groups, they fail to fy good and poor prognostic subsets within each stage. Since most patients present with early stage disease at diagnosis, a number of laboratory tests have been developed to try and predict the clinical course of these patients, most notably, immunoglobulin variable heavy chain somatic mutation status, CD38 expression, T-cell tyrosine kinase (ZAP-70) expression and cytogenetic WO 24264 PCT/G32012/051936 abnormalities. Unmutated IGHV genes, high CD38 expression, high ZAP-70 expression and the ce of 17p and 11q deletions are all associated with a poor prognosis. The exploitation of this sort of laboratory data to provide a prognostic assay is described in US 2008/0026383. However, none of these individual markers can provide definitive prognostic information alone and when used in ation offer only a reasonable prognostic tion.

Breast cancer is another very common tumour type in the western world. Breast s can be surgically removed but ts of the tumour can remain resulting in the reoccurrence of the disease. Patients therefore have adjuvant treatments that have toxic side effects, and the suspicion is that many patients receive treatment that will not be beneficial to them. The usual approach is to tailor the aggressiveness of the chemotherapy to the risk of recurrence. As compared with standard chemotherapy, aggressive chemotherapy is associated with a greater benefit, but also with more acute and long-term toxic effects such as leukaemia and heart failure.

As with CLL, there is thus a requirement for markers that allow stication following surgery for breast cancer. Gene expression arrays have been employed to identify specific gene expression signatures that are indicative of prognosis; these provide hazard ratios of up to 3.4 for overall survival in node ve breast cancer patients. Gene expression arrays are amongst the best markers of prognostication currently available for Breast cancer.

Myelodysplastic mes (MDS) are a heterogeneous collection of disorders of the bone marrow haematopoietic stem cells characterised by disruption to haematopoiesis ultimately g to bone marrow failure. This condition was previously known as ‘pre-leukaemia' because one third of patients progress to acute myeloid leukaemia (AML). There is therefore a clinical need to distinguish patients that progress to AML, and thus may require therapy from those that manifest a more benign form of the disease. Like CLL, MDS is characterised by large-scale unbalanced chromosomal rearrangements; these types of rearrangements are consistent with telomere dysfunction. rmore, there is evidence of telomere n in MDS and that mutation in the telomerase RNA components can confer MDS in children.

It follows from the above, that there is a range of diseases for which relatively early stage prognostication would be ageous. Moreover, many of these diseases are characterised by genetic abnormalities and, specifically telomere shortening.

These diseases include mer's disease‘, brain infarction‘, heart disease1, chronic HIV infection1, chronic hepatitis‘, skin diseases‘, chronic inflammatory bowel disese1 including ulcerative colitis, anaemia‘, atherosclerosis", Barrett's oesophagus and cancers1 including pre-cancerous conditions. The invention therefore has ation to all of these diseases. res are nucleoprotein structures composed of repetitive DNA sequences that cap the ends of linear eukaryotic chromosomes, protecting them from deterioration or fusion with adjacent chromosomes. During replication of DNA, the ends of chromosomes cannot be processed, and as a result during cell on the chromosome ends would be lost; res however prevent this by lves being consumed during each stage of cell division, essentially ‘capping’ the chromosome. re ends are, however, maintained in certain cell types such as germ cells, stem cells and certain white blood cells, by the reverse transcriptase telomerase that catalyses the RNA templated addition of telomere repeats.

Telomere length is a key determinant of telomeric function and it has been shown that short dysfunctional telomeres can drive genomic instability and igenesis in mouse models. Furthermore, deregulation of telomerase has been shown to drive oncogenesis. onally, the loss of res in somatic cells has been linked to ative senescence preventing genomic instability and cancer. Conversely, it has also been shown that malignant cells can bypass this ence and become immortalised by telomere extension by aberrant activation of telomerase.

Consistent with the role of re biology in tumour progression, there is now a ntial body of evidence indicating that telomere length can provide prognostic information in many human malignancies including CLL”. However, there is a lack of resolution in the currently available technologies and this has hampered progress in translating telomeric assays into clinical practice. For example, a putative role of telomere dysfunction during the progression of breast cancer has been shown,10 and low-resolution telomere length has been shown to provide limited prognostic information 11,12 A key problem with these technologies is that they are based on PCT/G32012/051936 hybridisation of DNA probes to telomere repeat units. Consequently, as telomeres get r there is less probe target, and thus short telomeres are not detectable13'14. This is important because it is the shortest telomeres that become dysfunctional and are subject to fusion, g c instability that can drive the progression of human cancers15'17. Q-PCR-based methods have also been described for the estimation of telomere repeat content (W0 2004068110US), these allow fer high hput analysis. However the linearity of these methods for the detection of short telomeres (< 4 kb) has not been established”, this, d with the reported high CV values of up to 28%, renders the Q-PCR s inappropriate for the detection of short res and using this information as a prognostic tool for al decision making19. Hitherto, telomere analysis using existing low-resolution ques is not a sufficiently informative prognostic marker.

To address this problem, we have previously developed single-molecule technologies that allow us to detect the presence of critically shortened resm'21 and to characterise telomere end-end fusions‘s'". Single telomere length analysis (STELA) allows complete resolution of telomere lengths at specific chromosome ends, including telomeres in the length range in which telomere end- end fusions can occur‘e'zo. It therefore permits detection of short telomeres that are potentially dysfunctional and capable of fusion. In part of this study the Xpr telomere was chosen for use in STELA because in contrast to 13q, 6q, 17p and 11q, there is no evidence to implicate the loss of this telomere in the ogy of CLL in particular. Furthermore our previous data indicate that the Xpr telomere length is representative of the genome-wide telomere lengthzo'”, and that telomerase- expressing cells can homogenise telomere lengths at different chromosome ends15'23. Using these tools, we have demonstrated a link between short telomeres, telomere end-end fusion events and genomic instability in diseases such as, but not d to, CLL breast cancer and MDS.

In our investigations, we have used telomere length and fusion analysis to provide a definition of telomere dysfunction and then we have used this as a prognostic tool.

Specifically, we have identified the longest mean telomere length at which telomere end-end fusion events can be detected for a selected chromosome, examples are shown in Table 1. Using this upper limit for fusion event detection we have been able to show that the mean telomere length in the fusogenic range (i.e. s the upper limit) provides a biological parameter that is highly prognostic for at least one of the following: time to first treatment, response to treatment or overall survival.

Furthermore, this biological ter can also be used to provide remarkable stic tion in early stage e patients in terms of time to first treatment, response to treatment or overall survival; indeed, patients in the longer telomere subset showed an l survival rate of 96% at 10 years. The longest mean telomere length at which telomere end-end fusion events can be ed therefore represents an indication of the mean telomere length at which telomeres become dysfunctional and capable of fusion. Knowledge of the length of an individual's telomeres and so the likelihood of d fusion events enables one to predict where the individual is placed with respect to disease progression and so ensures the individual receives treatment commensurate with their requirements; no less and no more. Further, the test to assess the length of an individual's telomeres can be repeated periodically to monitor disease progression.

We have been able to show that by ng a telomere length threshold based on telomere dysfunction, we are surprisingly able to transform the prognostic power of telomere length analysis. Thus in contrast to us reports using low-resolution telomere length analysis (i.e. those methods described above that measure telomere length at 4kb and above), our data indicate that high-resolution telomere length analysis (i.e. using, e.g. the STELA method, or any other method which can measure the full range of telomere length from one TTAGGG repeat to over 25kb of telomere length) d with a definition of telomere ction or a knowledge of our biological parameter, is sufficient for accurate prognostication in various diseases characterised by telomere shortening, including cancers.

Statements of Invention According to a first aspect of the invention there is provided a prognostic method for determining the ssion of a disease ing or terised by telomere shortening comprising: i) using high-resolution telomere length analysis to determine the longest mean telomere length at which telomere end-end fusion events can be detected in W0 2013/024264 2012/051936 samples of tissue from a number of individuals presenting with the same disease, in order to identify a threshold figure that represents an indication of the mean telomere length at which telomeres become dysfunctional and capable of fusion ; ii) determining the prognostic mean telomere length of samples of tissue from a number of individuals presenting with said disease, by taking those samples whose mean telomere length is less than said threshold and averaging the mean telomere length of those s; iii) determining the mean test telomere length of a sample taken from a patient suspected of having or presenting with said disease and. where said mean test telomere length is less than said prognostic mean telomere length, concluding time to first treatment is poor and/or response to treatment is poor and/or overall survival is poor; or iv) determining the mean test telomere length of a sample taken from a patient suspected of having or presenting with said disease and, where said mean test telomere length is greater than said prognostic mean telomere length, concluding time to first treatment is good and/or response to treatment is good and/or overall survival is good.

The ion ore involves the identification of a specific methodology that permits al telomeric parameters to be defined for a particular disease or, typically, malignancy. These parameters are the upper telomeric threshold for end- end fusion events, as in i) above, and a subsequent prognostic mean telomere length below the said old or in the fusogenic range, as in ii) above. Further, the invention also involves an analysis of patient telomere distribution, as in iii) or iv) above, and by ng this to the determined threshold and said prognostic mean, the invention predicts whether a t will require treatment and it also predicts progression-free or overall al of each t at the time the method is undertaken.

In a preferred method of the invention said fusion event in part i) above is verified as being such by direct DNA sequence analysis before the data relating to same is included in the method.

W0 2013/024264 Additionally or alternatively, in a further preferred method of the invention, said prognostic mean telomere length of a sample of tissue from a number of individuals presenting with said disease is determined by taking those samples that exhibit telomere fusion and averaging the mean telomere length of those samples. This preferred method therefore includes samples whose mean re length is less than said threshold and also s whose mean telomere length is greater than said threshold but, regardless of this fact, only samples exhibiting fusion are used to te an average telomere length. As those skilled in the art will appreciate, the fact that the method can be worked using this additional or alternative set of s indicates that any telomere length below said threshold is prognostic; the mean thereof particularly so.

In a further preferred method of the invention said e including or characterised by re shortening comprises a e where telomeres are shortened, as herein described, ularly where telomerase has reduced activity stically significant at the P<0.05 level) having regard to the e activity in immortalsied cell lines, and most preferably comprises one or more of the ing diseases: ageing, alzheimer’s disease; brain infarction; heart disease; c HIV infection; chronic hepatitis; skin diseases; chronic inflammatory bowel disease; ulcerative colitis; anaemia; atherosclerosis; Barrett's oesophagus; and , including pre- cancerous conditions.

Preferably said cancer is either a haematological malignancy or a solid tumour.

Yet more preferably said cancer is CLL, MDS or breast cancer.

Yet more preferably, said telomere length at which telomere end-end fusion events can be detected is, ideally but not necessarily, determined for a selected single chromosome. Examples of chromosomes on which this analysis has been undertaken are shown in Table 1 along with the value of the upper limit for end-end fusion detection for each chromosome. Using five examples we have shown that the upper limit for detecting end-end fusion events in different chromosomes is very similar i.e. between 3.81 and 5.01kb. The mean is 4.52kb with a standard deviation of only 0.46kb. Similarly, we have also shown that the mean telomere length in the PCT/G32012/051936 fusogenic range for these five somes is also very similar i.e. between 2.26 and 3.01 kb. The mean is 2.69kb with a standard ion of only 0.30kb.

In an alternative preferred method of the invention, said telomere length at which telomere d fusion events can be detected is determined for a number of ent chromosomes. Indeed, any chromosome could be used that can be subjected to high-resolution telomere length analysis. In this instance, the average upper limit for detecting end-end fusion events in the different chromosomes is used in part i) above; and the e mean telomere length in the fusogenic range for these different chromosomes in part ii) above is also used.

In a preferred method of the invention, in the case where said disease is CLL, time to first treatment is poor means an individual has a median time to treatment of less than 2 years (La. 1.84 years) with a hazard ratio of 23.2 indicating that they are 23.2 times more likely to require ent in unit time than an individual with telomere length above the threshold. Response to treatment is poor means a median time from first treatment to death of less than 5 years (i.e. 4.1 years) with a hazard ratio of 6.4 and overall survival is poor means a median survival time from diagnosis of less than 8 years (i.e. 7.49 years) with a hazard ratio of 71 .3.

In a preferred method of the invention, in the case where said disease is CLL, time to first treatment is good means an individual will not need treatment and can be monitored conventionally; and response to treatment is good means that the mean time to treatment will not be reached within 10 years; and overall survival is good means that the median survival is greater than 10 years with 96% of the cohort surviving to this censor point and can be monitored conventionally.

In a preferred method of the invention, in the case where said disease is MDS, overall al is poor means a median survival time from diagnosis of less than 1.5 years (i.e. 1.15 years) with a hazard ratio of 9.5.

In a preferred method of the invention, in the case where said disease is MDS, overall al is good means that the median survival is 4.9 years and can be monitored conventionally.

PCT/G32012/051936 In a preferred method of the invention, in the case where said disease is breast cancer, overall survival is poor means a median survival time of less than 1 year (i.e. 0.95 years) with a hazard ratio of 87080.

In a preferred method of the invention, in the case where said disease is breast cancer, overall survival is good means that the median survival is greater than 6 years and can be monitored conventionally.

According to a second aspect of the invention there is ed a prognostic method for determining the ssion of a disease including or characterized by telomere shortening comprising: i) using high-resolution telomere length analysis to determine the stic mean telomere length of samples of tissue from a number of individuals presenting with said disease, whose mean telomere length is less than a 4.52 kb telomere length threshold at which telomere d fusion events can be detected in said cancerous disease, by taking those samples whose mean telomere length is less than said threshold and averaging the mean telomere length of those samples; ii) determining the mean test re length of a sample taken from a t suspected of having or presenting with said disease and, where said mean test telomere length is less than said prognostic mean telomere length, concluding the time to first treatment is poor and/or the se to treatment is poor and/or overall survival is poor; or iii) determining the mean test telomere length of a sample taken from a patient suspected of having or presenting with said disease and, where said mean test telomere length is greater than said prognostic mean telomere length, concluding time to first treatment is good and/or response to treatment is good and/or l survival is good.

In this second embodiment of the invention, preferably, said prognostic mean telomere length is determined using a 4.06kb threshold (i.e. 4.52 — 0.46kb) or a 4.98kb threshold (is. 4.52 + ) at which telomere end-end fusion events can be detected.

PCT/G32012l051936 In a preferred embodiment of the second aspect of the invention said disease is cancer and, typically, said cancer is CLL, breast cancer or MDS and, ideally, said prognostic mean telomere length value of 2.26kb is used for CLL and breast cancer and said stic mean re length value of 2.5kb is used for MDS.

Yet more preferably, in this second aspect of the invention said telomere length at which telomere end-end fusion events can be detected is determined for a number of chromosomes. Ideally, the chromosomes are Xpr, 17p, 2p, 16p and 18q, although any other combination of somes may be used and their average upper threshold at which telomere end-end fusion events can be detected is used in the above method.

According to a third aspect of the invention there is provided a prognostic method for determining the progression of a e including or characterized by telomere shortening comprising: 1. determining the mean test telomere length of a sample taken from a patient suspected of having or ting with said disease and, where said mean test telomere length is less than a prognostic mean telomere length of 2.69kb, concluding the time to first treatment is poor and/or the se to treatment is poor and/or overall survival is poor; or 2. determining the mean test telomere length of a sample taken from a patient suspected of having or presenting with said disease and, where said mean test telomere length is greater than a prognostic mean telomere length of 2.69kb, concluding the time to first treatment is good and/or the response to ent is good and/or overall survival is good.

In this third embodiment of the invention, preferably, said prognostic mean telomere length is either 2.39kb (i.e. 2.69 — 0.3kb) or 2.99kb (i.e. 2.69 + 0.3kb).

In a preferred embodiment of the third aspect of the invention said e is a haematological , and typically said cancer is CLL or MDS and, more ideally still, said prognostic mean telomere length is 2.26kb for the former and 2.5kb for the Iaflen In a preferred embodiment of the third aspect of the invention said disease is breast cancer and, more ideally still, said prognostic mean telomere length is 2.26kb.

Yet more ably, in this third aspect of the invention said prognostic mean telomere length is determined for a number of chromosomes. Ideally, the chromosomes are Xpr, 17p, 2p, 16p and 18q, although any other combination of chromosomes may be used and their average prognostic mean telomere length is used in the above method.

In a related ment there is provided one or more, ing combinations thereof, of the primers described herein.

In another related embodiment there is provided a treatment regimen including or comprising said afore prognostic method according to any aspect or embodiment of the invention.

In the claims which follow and in the preceding description of the invention, except where the context requires othen/vise due to express language or ary implication, the word ises”, or variations such as “comprises” or “comprising” is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

All nces, including any patent or patent application, cited in this specification are hereby incorporated by reference. No ion is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general dge in the art.

Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

Other features of the present invention will become apparent from the following examples. Generally ng, the invention extends to any novel one, or any novel PCT/G320121051936 combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus. es. integers, characteristies. compounds or chemical moieties described in conjunction with a particular aspect. embodiment or example of the invention are to be tood to be applicable to any other aspect. ment or example described herein. unless incompatible therewith.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

The invention will now be described by way of example only with reference to the following tables and figures: Table-1 shows the longest mean telomere length at which telomere end-end fusion events can be detected for a range of chromosomes. including the mean thereof and the prognostic mean telomere length for each one of said chromosomes, including the mean thereof.

Table-2 shows a comparison of prognostic factors in univariate analysis, in terms of time to first treatment and overall survival.

Table-3 shows the clinical teristics of the 184 CLL patiem cohort. 4 shows the analysis of concordant datasets combining telomere length analysis with known prognostic s.

Figure-1 defines the telomeric parameters for prognosis in CLL. [A] An example ofSTELA at the Xpr telomere in 12 CLL patients in which fusion was, or was not ed. Mean and standard deviation are displayed below and the means ghted in red on the gel image. [B] Examples of fusion analysis in 4 CLL patients. [C] Examples of the DNAsequence of the fusion events highlighted in panel B. Arrows indicate the fusion junction. together with the participating - telomere and the deletion from the start of the respective res. Homology between the participating res is underlined. [D] Mean Xpr re length data plotted as a function of Binet staging. Black squares indicate those that were not tested for , empty squares those that were negative and marked squares those that were positive for fusion events. Panel E shows telomere length data from the whole cohort, together with those that were positive for fusion events. The longest mean Xpr telomere (3.81 kb) in which fusion was detected is indicated with a dashed line and mean Xpr telomere length of the samples in which fusion was detected was 2.26kb.

Figure-2. Mean telomere length is prognostic in CLL. Panels A and B show Kaplan Meier cunles from the entire cohort for time to first treatment upper graph and l survival lower graph. P values. Hazard Ratio (HR) are indicated on the plots together with numbers in each arm.

Figure-3 shows that telomere length. as defined by , is highly prognostic in CLL. [A-B. E] Kaplan Meier curves from the entire cohort, {or time to first treatment and overall survival. P- values and Hazard Ratio (HR) are indicated on the plots, together with numbers in each arm.

[C-D] Kaplan Mei'er curves for the Binet stage A only . [F-G] Recursive partitioning of the data set shows 2.26kb is the optimal re threshold as a prognostic tool for defining survival in the whole data set, and in the 2 population s.

Figure-4. Panel A shows mean 17p telomere length data plotted as a function of Binet staging.

Black squares indicate those that were not tested for fusion. empty squares those that were negative and marked squares those that were positive for fusion events. Panel B shows telomere length data from the whole cohort. together with those that were positive for fusion events. The longest mean Xpr telomere (4.81 kb) in which fusion was detected is indicated with a dashed line and denotes the upper limit of the fusogenic range for the 17p telomere. The mean telomere length of the samples in which fusions could be detected was . Panels C and D show Kaplan Meier curves for time to first treatment and overall al tively based on a cut-off of 2.5kb derived from recursive partitioning of the data. Panels E and F show Kaplan Meier curves for time to first treatment and overall survival respectively for stage A patients only based on a cut-off of 2.5kb derived from recursive partitioning of the data. Panel G shows a plot of mean telomere length of the 17p telomere versus hazard ratios for overall survival. Recursive partitioning illustrates that 2.5kb is the l threshold for defining prognosis using this telomere.

PCT/GBZ012I051936 -5. shows that telomere length is superior to other known stic parameters. Kaplan Meier curves with telomere length together with cytogenetics [A-B], IGHV status [C—D], CD38 status [E-F] and ZAP-70 status [G-H] as a function of both time to first treatment and overall survival.

Figure-6. Shows that the telomere threshold of 2.26kb, derived from the Xpr chromosome, is highly stic for CLL patient se to treatment. Kaplan Meier curves for a subset of patients with CLL that received treatment (n = 75).

Survival time was calculated from time of first treatment. P-values and Hazard Ratio (HR) are indicated on the plots, together with numbers in each arm.

Figure-7. Shows that telomere length, as defined by , is also prognostic in breast cancer. [A-D] Kaplan Meier curves from the entire cohort, for overall survival.

P-values and Hazard Ratio (HR) are indicated on the plots, together with numbers in each arm. [E] Recursive partitioning of the data set shows that 2.26kb is the l telomere threshold as a prognostic tool for defining survival in the whole data set.

Figure-8 MDS figure shows that the 2.26kb telomere threshold offers limited prognostic power in MDS. [A-D] Kaplan Meier curves from the entire cohort, for overall survival. P-values and Hazard Ratio (HR) are indicated on the plots, together with numbers in each arm. D shows the 2.5kb telomere threshold offers better stic power in MDS. [E] Recursive partitioning of the data set shows that 2.5kb is the l telomere threshold as a prognostic tool for defining survival in the whole data set.

METHODS CLL Patients Peripheral blood samples from 184 CLL consenting patients, in ance with the Declaration of Helsinki and as approved by the South East Wales local research ethics committee (LREC# 02/4806). CLL was defined by clinical criteria as well as cellular morphology, and also the co—expression of CD19 and CD5 in lymphocytes W0 2013/024264 PCT/G32012l051936 simultaneously displaying restriction of light-chain rearrangement. Comprehensive clinical information was available for all patients with a median follow-up of 5.8 years.

All of the samples were collected at, or close to, the time of diagnosis from two centers, Cardiff and Birmingham, and staging was based on the Binet classification system“. The clinical characteristics of the CLL patient cohort are presented in Table-2.

Breast cancer Patients Genomic DNA, together with clinical follow up data, from a panel of 28 invasive breast ductal carcinomas was obtained from the Wales Cancer bank, under approval from the Wales MREC.

MDS Patients Bone marrow samples were ed from 63 patients diagnosed with ysplastic syndrome (MDS), as classified according to the French-American- British system. Of these, 40 patients were male and 23 were female, with a mean age at diagnosis of 67.5 years; the median follow-up for the cohort was 5.6 years.

IPSS criteria were available for 55/63 patients with 15 high, 20 intermediate and 20 low.

Isolation ofperipheral blood mononuclear cells from CLL ts Peripheral blood mononuclear cells (PBMCs) were ed from EDTA venous blood of the 184 CLL patients by density centrifugation using Fiooll-Hypaque (lnvitrogen).

B—cells were subsequently positively isolated using CD19-labeled Dynabeads (lnvitrogen)25. Cells were stored at -20°C as dry pellets prior to DNA extraction.

DNA extraction and PCR DNA was extracted from human cells using standard proteinase K, RNase A, phenol/chloroform protocolsze. For telomere length is at the Xpr, 17p, 2p, 16p and 18q res, we used a modification of the single telomere length analysis (STELA) assay as previously described‘s'zo. Briefly, genomic DNA was solubilized by dilution in 10mM CI (pH 7.5), fied by using Hoechst 33258 fluorometry (BioRad, Hercules, USA), and diluted to l in 10mM Tris-HCl (pH 7.5). DNA (10ng) was further diluted to 250 pg/pl in a volume of 40ul, containing Telorette2 linker (1pM) and Tris-HCI (1mM; pH 7.5). Multiple PCR reactions ally 6 reactions per sample) were carried out for each test DNA, in 10ul volumes. The reaction mixture consisted of DNA (250pg), telomere-adjacent and Teltail primers (0.5uM), Tris-HCI (75mM; pH8.8), (NH4)2SO4 (25mM), 0.01% Tween-20, MgCI2 ), and 0.5 U of Taq (ABGene, Epsom, UK) and Pwo polymerase (Roche Molecular Biochemicals, Lewes, UK) in a 10:1 ratio. The reactions were cycled with an MJ FTC-225 thermocycler (MJ research, Watertown, USA). The DNA fragments were resolved by 0.5% TAE agarose gel electrophoresis, and detected by two separate Southern izations, with random-primed a-33P labeled (Amersham Biosciences, Little Chalfont, UK) TTAGGG repeat probe and a telomere-adjacent probe, together with a probe to detect the 1kb (Stratagene, La Jolla, USA) and 2.5 kb (BioRad) molecular weight marker. The hybridized fragments were detected by phosphorimaging with a Molecular Dynamics Storm 860 orimager (Amersham Biosciences, Little Chalfont, UK). The molecular weights of the DNA fragments were calculated using the ix 1D quantifier (Nonlinear Dynamics, Newcastle-upon-Tyne, UK).

Telomere fusion was ed using the usly described single molecule telomere fusion assaysm'17. PCR reactions containing 100ng of DNA were performed, each containing the XprM, 17p6 and 21q1 PCR primers. Fusion molecules were detected, and the ncies quantified by rn blotting and hybridization with the Xpr telomere-adjacent probes as described usly”.

In order to determine the chromosomes participating in the fusion events for uent sequence characterization, further hybridisations were undertaken with the 17p and 21q telomere adjacent probes; the 21q probe yields onal non-specific products and thus was not used for quantification. Any fusion products were then re-amplified for direct sequence analysis using nested PCR primers (XprO, 17p? and 21qseq1).

The oligonucleotides utilised were: XprM ([SEQ ID NO: 1] 5'- ACCAGG'I‘I'TTCCAGTGTGTT—3'), 1 7p6 ([8E0 lD NO: 2] 5'- GGCTGAACTATAGCCTCTGC-B'), 21 q1 ([8E0 ID NO: 3] 5'- CTTGGTGTCGAGAGAGGTAG-3') for fusion PCR; XprO ([SEQ ID NO: 4] 5'- CCTGTAACGCTGTTAGGTAC-S'), 1 7p? ([8EQ ID NO: 5] 5'- CCTGGCATGGTATTGACATG-3'), 21qseq1 ([SEQ ID NO: 6] 5'- TGGTCTTATACACTGTG'I'I'C -3') for re-amplification of fusion products; 21qseq1 ([SEQ ID NO: 6] 5'-TGGTCTTATACACTGTGTTC -3'), 21 qseq1rev ([SEQ ID NO: 7] 5'-AGCTAGCTATCTACTCTAACAGAGC-3'), XprO ([SEQ ID NO: 4] 5'- CCTGTAACGCTGTTAGGTAC-3'), XprB2 ([SEQ ID NO: 8, 9] 5'- TCTGAAAGTGGACC(A/T)ATCAG-3'), 17p7 ([SEQ ID NO: 5] 5'- CCTGGCATGGTA'I'I'GACATG-3'), 17pseq3 ([SEQ ID NO: 10] 5'- CTGTCCTCAACAAGT-S') to generate hybridisation probes for fusion analysis.

Primers that can be used for STELA analysis (the ones that are typically used emboldened): XprEZ (SEQ ID NO : 11) TTGTCTCAGGGTCCTAGTG Xpr-427AI415T (SEQ ID NO: 12) GGTTATCAACCAGGTGCTCT 7GI415C (SEQ ID NO: 13) GGTTATCGACCAGGTGCTCC Xprins (SEQ ID NO: 14) TGGAATTGGTGGGTT XprdeI (SEQ ID NO: 15) CCTAGTGTGTCTGGAATTGGTTC XprM (SEQ ID NO: 1) ACCAGGT'ITI'CCAGTGTGTT XprC (SEQ ID NO: 16) CAGGGACCGGGACAAATAGAC XprO (SEQ ID NO: 4) CCTGTAACGCTGTTAGGTAC 17psevq1rev (SEQ ID No: 17) ACGGATTGCTTTGTGTAC 17p6 (SEQ ID NO: 2) GGCTGAACTATAGCCTCTGC 17p7 (SEQ ID NO: 5) CCTGGCATGGTATTGACATG 16prev1 (SEQ ID NO: 18) GTGAATAATCAAGGTCAGAGCA 18qrev4 (SEQ ID NO: 19) CCTGTGGGTCTAAAACCAGAAGG 2p2 (SEQ ID NO: 20) GAGCTGCG'ITTTGCTGAGCAC 11q13B (SEQ ID NO: 21) TTGGAGGCACGGCCTTCG 12q-197A (SEQ ID NO: 22) GGGAGATCCACACCGTAGCA 12q-550C (SEQ ID NO: 23) ACAGCCTTTTGGGGTACCGC WO 24264 Statistical methods Statistical analysis was carried out using Prism 3.0 (Graphpad) and SAS version 9.1.3 software (SAS Institute).

The relationship between telomere length. known prognostic s, time to first ent (TTFT) and overall survival (03) were explored through Wilcoxon rank sum tests for the categorical variables Binet stage, 6038, ZAP-70, lGHV gene mutation status. flZ-microglobulin and FlSH cytogenetics. Unstratified univariate comparisons of survival between the prognostic subsets were conducted with the log-rank test, with survival data displayed using Kaplan-Meier curves. Multivariate analysis, which adjusted for other prognostic features. was performed using forward selection to define cant co-variables with Cox regression. A P-value < 0.05 was considered significant.

RESULTS Telumere length and fusion is We analyzed the telomere length distribution in 184 CLL patients using single telomere length is ) at the Xpr telomere (Figure 1A). Given that we have previously shown that telomere end-end fusion events can be detected in CLL patients with short telomeres“. we systematically looked for telomere fusions in the CLL samples with the shortest mean re lengths (n = 88). We only considered a fusion event to be bone tide when it could be fully characterized by direct DNA sequence analysis (Figure 13, 10). Telcmere s were detectable in samples derived from all Binet stages, suggesting that they are not merely a characteristic of advanced disease e 1D; fusions shown as marked squares). r, fusions were only detected in samples with a mean telomere length of £3.81kb. We therefore used this telomere length as a old to define the upper limit of the ‘fusogenic' range for our cohort using this chromosome. Figure 1E shows that 981184 (53.3%) of the CLL samples had a mean Xpr telomere length equal or less than 3.81kb with a mean fusogenic telomere length of 2.26kb. We therefore used 2.26kb as a way of defining. two subsets of CLL patient samples in our cohort and determined the prognostic value of this mean telomere length threshold in our cohort. A total of 33I184 (17.9%) of the samples had a mean telomere length 52.26kb.

PCT/G32012/051936 Telomere dysfunction is highly prognostic in CLL In keeping with previous studies, mean telomere length was prognostic in our cohort of patients for TTFT (P<0.001; HR=5.5) and OS (p=0.0017; HR4.2) (Figure 2).

However, categorization of the samples based on telomere dysfunction ($2.26kb telomere length of the Xpr telomere) revealed remarkably enhanced prognostic discrimination. Figure 3A and 3B show that a mean telomere length $2.26kb was highly prognostic for TTFT and OS. The median 'ITFT was 1.8 years (P<0.0001; HR = 23.2) and the median 08 was 7.5 years (P<0.0001; HR = 71.3). In contrast, the median TTFT and 08 were not reached in the longer telomere subset. ularly striking was the impact of telomere length on OS in our cohort; the Kaplan Meier curve for patients with >2.26kb telomere length showed almost no erosion over the year follow-up ; 98% survival at 5 years and 96% al at 10 years.

Whereas, only 36% of the short telomere group was alive at the 10-year censor point indicating that patients with $2.26kb were more than 70 times more likely to die in unit time. These data are summarised in 2.

Stage A patients with short res have more aggressive disease Given that the majority of CLL patients present with early stage disease and this group represent the greatest nge in terms of prognostication, we performed a subset is of only the Binet stage A patients. 130/184 (70.6%) of our cohort was Binet stage A at diagnosis of which 15 (11.5%) had 52.26kb telomere length for the Xpr telomere. Figures 3C and 3D show the prognostic impact of short telomeres in early stage disease. The median TTFT was 2.1 years (P<0.0001; HR = 33.0) and the median 08 was 9.0 years (P<0.0001; HR = 994.2). Once again, the median TTFT and OS were not reached in the longer telomere group. The remarkable hazard ratio for OS suggests that these patients are almost 1000 times more likely to succumb to their disease in unit time than patients with longer telomeres. Once again, the superior Kaplan Meier curve revealed that patients with longer telomeres had a survival rate of 96% at 10 years. ion of the dataset to 144 Stage A patients, provided r verification that the specific telomere length of 2.26 kb ed the maximal prognostic power for this assay in CLL and the HR for overall survival increased to 1353 (Figure 3E).

PCT/G32012/051936 ive partitioning identifies the 2.26kb threshold as most prognostic for survival Although we had experimentally ined the telomere length for telomere dysfunction in CLL and shown that this was highly prognostic, we wanted to establish if this represented the optimal telomere length cutoff for predicting survival in our cohort. By performing ive partitioning on our data set, we found 2.26kb represented the optimum telomere , and was the most prognostic threshold for the total cohort and the Stage A cohort (Figure 3E). Given that our cohort was made of samples derived from two different centers (Cardiff and Birmingham), we repeated the analysis in these two separate populations and derived essentially the same result (Figure 3F). This ch provides further circumstantial evidence that 2.26kb represents the biological limit of telomere stability and confirms the clinical ance of this mean fusogenic telomere length in CLL.

We considered that this mean fusogenic telomere length may be conserved at other chromosome ends and thus we analyzed telomere length at 17p (Figure 4A) in 149/184 (81%) of the patient . The mean 17p telomere length of the samples in which we could detect s was similar to that observed at Xpr (2.57kb, .79, P = 0.21; Figure 4B). Recursive partitioning revealed the optimal telomere length for determining prognosis was 2.5kb; this was highly prognostic in the whole cohort (OS P<0.0001, HR = 72) and stage A patients (OS P = 0.009, HR = 71, Figure 4C-G). re length is or to other prognostic parameters We next investigated the impact of dysfunctional telomeres on other known prognostic markers in CLL, including cytogenetics, IGHV mutation status, CD38 expression, ZAP-70 expression and Beta-2 microglobulin (BZM). The combined analysis of telomere length with FISH cytogenetics, IGHV mutation status, CD38 expression, ZAP-70 expression are shown in Figure 5. As shown, short telomere length defines poor prognostic subsets of patients within cytogenetic risk groups, IGHV unmutated and mutated , CD38+ and CD38' groups, ZAP-70+ and ZAP- 70' groups and [32M high and low groups, in terms of 'I'I'FT and OS. ing these markers with telomere length enhanced the prognostic power still further; for W0 24264 PCT/G32012/051936 example, the analysis of the concordant datasets revealed that high CD38 expression in ction with telomere length b yielded a HR of 2915 (P<0.0001, Table-4).

Telomere length is the dominant co-variable in multivariate analysis In multivariate analysis fonNard selection identified telomere dysfunction (52.26kb) as the most significant parameter for TTFT (HR = 4.2; CI 1.9-8.8, P = 0.0002) and OS (HR = 10.9; CI 3.8-312, P<0.0001). Only IGHV mutation status and Binet stage retained ndent prognostic significance as co-variables in the model for TTFT and only CD38 in terms of OS. It is of ular interest that IGHV mutation status and ‘high-risk’ cytogenetics were not independently prognostic in terms of OS. To our knowledge, this is the first time that these parameters have failed to prove significant for OS in this disease.

Telomere length defines response to ent in CLL Given that we have shown that telomere length provides powerful prognostic ation in CLL, we further considered that telomere length may also provide information about the ability of patients to respond to treatment. We therefore undertook a subset analysis (n=75) of our CLL patient cohort for those that had received treatment. Telomere length was highly prognostic for response to treatment with a HR of 6.4 (P = 0.0002) (Figure-6).

Telomeric ters defined in CLL are prognostic in other indications.

We examined a cohort of 28 patients with invasive ductal carcinoma of the breast.

We analyzed Xpr telomere length using STELA and categorized the patients based on the 2.26kb telomere length cutoff defined in CLL. Despite a limited follow up period of 4.6 years, the 2.26kb mean fusogenic telomere length ed remarkable levels of prognostication for l survival in this disease with a hazard ratio of 112 (P = 0.0056), and a median al in the poor prognostic group of 301 days (Figure-7A-C). Expansion of the dataset to 120 breast cancer patients, provided further verification that the specific telomere length of 2.26 kb ed the maximal prognostic power for this assay in breast cancer and the HR for overall survival increased to 87080 (Figure 7D).

PCT/(9320121051936 As with CLL, ive partitioning of the Breast Cancer Cohort data showed that the optimum telomere length as defined by HR was 2.26kb (Figure-7E).

We also examined telomere length in MDS using STELA and used the mean fusogenic telomere length defined in CLL to provide prognostic information in MDS.

We analysed a panel of 63 MDS patients for which we had survival data. The 2.26kb mean fusogenic telomere length as defined in CLL, ed some prognostic power in MDS with a HR of 4.7 (P = 0.09) for overall survival (Figure—8A-C). Unlike the CLL and breast cancer samples, the MDS samples were not purified and contained varying unidentified proportions of unaffected cells. We considered that the presence of cted normal cells would skew the optimal telomere length threshold for prognostication in this cohort. This was nt from the recursive partitioning, where the l telomere length was 2.5kb (HR = 9.5, P = 0.026) a difference of just 240 bp (Figure-8E). Expansion of the dataset to 78 MDS patients provided further verification that the ic telomere length of 2.5 kb provided the maximal prognostic power of this assay in MDS and the HR for overall survival sed to .45 (Figure 7D). Purification of MDS cells using CD34 may improve the accuracy of telomere-based prognostication in MDS.

Summary The main gs of this study can be summarised as follows: Telomere length analysis, as defined by telomere dysfunction, provides a highly prognostic tool in human diseases, such as CLL and other human malignancies, permitting considerable discrimination for clinical outcome following treatment.

Prognostic power should enable clinicians to confidently predict the clinical course of these geneous es.

Moreover, telomere dysfunction provides remarkable prognostic resolution in early disease stage.

Only telomeres in the lower portion of the length distribution profile have the propensity for end-end fusion; using the Xpr chromosome a telomere length of $2.26kb is a mean fusogenic telomere length for telomere ction in a primary human tumor, below which patients of human malignancies show poor prognostic W0 2013/024264 PCT/G32012/051936 outcome. Using a number of chromosomes a telomere length 52.69kb is a predictor for telomere dysfunction.

Patients with Xpr telomeres longer than 2.26kb have remarkably stable and indolent disease (98% of these patients were alive at 5 years and 96% at the 10-year censor point). tent re is in MDS and breast cancer shows that high-resolution telomere length analysis is likely to be highly prognostic in other haematological malignancies but antly also in solid tumours.

By applying a telomere length threshold based on telomere dysfunction, this transforms the prognostic power of re analysis into the most prognostic parameter ever described in both univariate and multivariate analysis.

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J Clin Oncol 2004;22:1807-14.

. Meeker AK, Argani P. Telomere ning occurs early during breast tumorigenesis: a cause of chromosome destabilization underlying malignant transformation? J Mammary Gland Biol Neoplasia 2004;9:285-96.

. Damle RN, alla FM, Ghiotto F, et al. Telomere length and telomerase activity delineate distinctive replicative features of the B-CLL ups defined by immunoglobulin V gene mutations. Blood 2004;103:375-82.

. Grabowski P, Hultdin M, Karlsson K, et al. Telomere length as a prognostic parameter in chronic lymphocytic leukemia with l reference to VH gene mutation status. Blood 2005;105:4807-12.

. Rossi D, Lobetti Bodoni C, Genuardi E, et al. Telomere length is an independent predictor of al, treatment requirement and Richter's syndrome transformation in chronic lymphocytic leukemia. Leukemia 2009;23:1062-72.

. Sellmann L, de Beer D, Bartels M, et al. Telomeres and prognosis in patients with chronic lymphocytic leukaemia. Int J Hematol 2011;93:74-82.

. Roos G, Krober A, Grabowski P, et al. Short telomeres are associated with genetic complexity, high risk genomic aberrations, and short survival in chronic lymphocytic leukemia. Blood 2008; 111:2246—52.

. Ricca l, Rocci A, Drandi D, et al. Telomere length identifies two different prognostic subgroups among VH-unmutated B-cell chronic lymphocytic leukemia patients. ia 2007; 21 05.

. Chin K, de Solorzano CO, Knowles D, et al. In situ analyses of genome ility in breast cancer. Nat Genet. 2004; 36:984-8. 11.Heaphy CM, Baumgartner KB, i M, Baumgartner RN, Griffith JK. Telomere DNA content predicts breast cancer-free survival al. Clin Cancer Res. 2007; 13:7037-43.

PCT/G32012/051936 12. Lu L, Zhang C, Zhu G, et al. Telomerase Expression and Telomere Length in Breast Cancer and their Associations with Adjuvant Treatment and Disease Outcome. Breast Cancer Res. 2011; 13:R56. 13.Aubert G, Hills M, Lansdorp PM. Telomere length measurement—Caveats and a critical assessment of the available technologies and tools. Mutat Res. 2011 Jun 8 [Epib ahead of print]. 14. Baird DM. New developments in telomere length is. Exp Gerontol. 2005; 40:363-8.

. Lin TT, Letsolo BT, Jones RE, et al. Telomere dysfunction and fusion during the progression of c cytic leukaemia: evidence for a telomere crisis. Blood 2010; 116:1899-907. 16. Capper R, Britt-Compton B, Tankimanova M, et al. The nature of telomere fusion and a tion of the critical telomere length in human cells. Genes Dev. 2007; 21 :2495-508. 17. Letsolo BT, Rowson J, Baird DM. Fusion of short telomeres in human cells is characterised by extensive deletion and microhomology and can result in x rearrangements.

Nucleic Acids Res. 2010; 38:1841-52. 18. Cawthon, R.M. Telomere measurement by quantitative PCR. Nucleic Acids Res. 2002; 30; 947. 19. Shen, J., Terry, M.B., Gurvich, l., Liao. Y.. Senie, RT. and Santella, R.M. Short telomere length and breast cancer risk: a study in sister sets. Cancer Res. 2007; 67: 5538-5544.

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. Baird DM, Rowson J, Wynford-Thomas D, Kipling D. ive allelic variation and ultrashort telomeres in senescent human cells. Nat Genet. 2003; 33203-7. 21. Britt-Compton B, Rowson J, Locke M, Mackenzie l, Kipling D, Baird DM. Structural stability and chromosome-specific telomere length is governed by cis-acting determinants in humans. Hum Mol Genet. 2006; 15:725-33. 22. Baird DM, Britt-Compton B, Rowson J, Amso NN, Gregory L, Kipling D. Telomere instability in the male gennline. Hum Mol Genet. 2006; 15:45-51. 23. Britt-Compton B, Capper R, Rowson J, Baird DM. Short res are preferentially elongated by telomerase in human cells. FEBS Lett 2009; 583:3076-80. 24. Binet JL, Auquier A, Dighiero G, et al. A new prognostic classification of chronic lymphocytic ia d from a multivariate survival analysis. Cancer 1981; 48: 198-206.

. Hewamana S, Alghazal S, Lin TT, et al. The NF-kappaB subunit Rel A is associated with in vitro al and clinical disease progression in c lymphocytic leukemia and represents a ing therapeutic target. Blood 2008; 111:4681-9. 26. Sambrook J, Fritsh EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd Edition ed.

New York: Cold spring harbour laboratory press; 1989.

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Claims (21)

1. . A stic method for determining the progression of a disease including or characterised by re shortening comprising: using high-resolution telomere length analysis to determine the longest mean telomere length at which telomere end-end fusion events can be detected in samples of tissue from a number of individuals presenting with the same e, in order to identify a threshold figure that represents an indication of the mean telomere length at which telomeres become dysfunctional and capable of fusion ; ii) determining the stic mean telomere length of samples of tissue from a number of individuals presenting with said disease, by taking those samples whose mean telomere length is less than said threshold and averaging the mean telomere length of those samples; iii) determining the mean test telomere length of a sample taken from a patient suspected of having or presenting with said disease and, where said mean test telomere length is less than said prognostic mean telomere length, concluding time to first treatment is poor and/or response to treatment is poor and/or overall al is poor; or iv) determining the mean test telomere length of a sample taken from a patient ted of having or ting with said disease and, where said mean test telomere length is r than said prognostic mean telomere length, concluding time to first treatment is good and/or response to ent is good and/or overall survival is good.
2. The method according to claim 1 wherein said fusion event in part i) above is verified as being such by direct DNA sequence analysis.
3. The method according to claim 1 or 2 wherein, additionally or alternatively, said prognostic mean telomere length of s of tissue from a number of individuals presenting with said disease is determined by taking those samples that exhibit telomere fusion and ing the mean telomere length of those samples.
4. . The method according to any preceding claim wherein said disease is one of the following diseases: aging, alzheimer's disease; brain infarction; heart disease; chronic HIV infection; chronic hepatitis; skin diseases; chronic matory bowel disease; tive colitis; a; atherosclerosis; Barrett’s oesophagus and cancer, including pre-cancerous conditions.
5. 5. The method according to claim 4 wherein said cancer is either a haematological malignancy or a solid tumour.
6. 6. The method according to claim 5 wherein said cancer is CLL, MDS or breast cancen
7. 7. The method according to any preceding claim wherein: said telomere length at which telomere end-end fusion events can be detected is determined for a single chromosome.
8. 8. The method according to any one of claims 1 to 6 wherein said telomere length at which telomere end-end fusion events can be detected is determined for a number of different chromosomes.
9. 9. The method according to claim 8 wherein the average upper limit for detecting end-end fusion events in ent chromosomes is used in part i) of claim 1 and the e mean re length in the fusogenic range for these different somes is used in part ii) of claim 1.
10. 10. A prognostic method for determining the progression of a e including or characterized by telomere shortening comprising: i) using high-resolution telomere length analysis to determine the prognostic mean telomere length of s of tissue from a number of individuals presenting with said disease, whose mean telomere length is less than a 4.52kb telomere length threshold at which telomere end-end fusion events can be detected in said cancerous disease, by taking those samples whose mean telomere length is less than said threshold and averaging the mean telomere length of those samples; ii) determining the mean test telomere length of a sample taken from a patient suspected of having or presenting with said disease and, where said mean test telomere length is less than said prognostic mean telomere length, ding the time to first treatment is poor and/or the response to treatment is poor and/or overall survival is poor; or iii) determining the mean test telomere length of a sample taken from a patient suspected of having or presenting with said disease and, where said mean test telomere length is greater than said prognostic mean telomere length, ding time to first treatment is good and/or se to treatment is good and/or overall survival is good.
11. 11. The method according to claim 10 wherein said disease is a cancer such as CLL, breast cancer or MDS.
12. 12. The method according to claim 11 wherein said prognostic mean telomere length is 2.26kb.
13. 13. The method according to claim 10 or 11 wherein said telomere length at which telomere end-end fusion events can be detected is determined for a number of different chromosomes.
14. 14. The method ing to claim 13 wherein the somes are Xpr, 17p, 2p, 16p and 18q.
15. 15. The method according to any one of claims 10 to 14 wherein in part i), onally or alternatively, said prognostic mean telomere length of samples of tissue from a number of individuals presenting with said disease is determined by taking those samples that exhibit telomere fusion and averaging the mean telomere length of those samples.
16. 16. A prognostic method for determining the progression of a disease including or characterized by telomere shortening comprising: i) determining the mean test re length of a sample taken from a patient suspected of having or presenting with said disease and, where said mean test telomere length is less than a prognostic mean telomere length of 2.69kb, concluding the time to first ent is poor and/or the response to treatment is poor and/or overall survival is poor; or ii) determining the mean test telomere length of a sample taken from a patient suspected of having or presenting with said disease and, where said mean test telomere length is greater than a prognostic mean telomere length of 2.69kb, concluding the time to first treatment is good and/or the response to treatment is good and/or overall survival is good.
17. 17. The method according to claim 16 wherein said disease is a haematological cancer such as CLL or MDS.
18. 18. The method according to claim 17 n said prognostic mean telomere length is 2.26kb.
19. 19. The method according to any one of claims 15 to 18 wherein said prognostic mean telomere length is determined for a number of different chromosomes.
20. 20. The method according to claim 19 wherein the chromosomes are Xpr, 17p, 2p, 16p and 18q.
21. 21. The method according to any one of claims 15 — 20 wherein in part i), additionally or alternatively, said prognostic mean telomere length of samples of tissue from a number of duals presenting with said disease is determined by taking those samples that exhibit telomere fusion and averaging the mean re length of those samples. @643P NO