WO2010142467A1 - Method of monitoring a disease caused by genomic translocation - Google Patents

Abstract

The present invention relates to methods of monitoring, diagnosing and gaining insights into the status and prognosis of, in particular, chronic myeloid leukaemia patients based on, for example quantitative analysis of genomic DNA of leukemic cells. It also extends to methods of treatment based on the results of the method.

Description

METHOD OF MONITORING A DISEASE CAUSED BY GENOMIC TRANSLOCATION

The present invention relates to methods of monitoring, diagnosing and gaining insights into the status and prognosis of a disease generated by or derived from a genomic translocation in a cell or cells of a patient, in particular a chronic myeloid leukaemia patient, based for example on quantitative analysis of genomic DNA of leukaemic cells. It also extends to methods of treatment based on the results of the method.

Chronic myeloid/myelogenous leukaemia (CML also known as chronic granulocytic leukaemia (CGL)) is a clonal malignant myeloproliferative disorder which originates in a single haematopoietic stem cell. The progeny of this abnormal stem cell, immature granulocytes

(neutrophils, eosinophils, and basophils), proliferate over months or years. The increased number of white blood cells in the bone marrow may result in low numbers of normal cells including red blood cells.

Thus chronic myeloid leukaemia is characterized by the increased and unregulated growth of predominantly myeloid cells in the bone marrow and the accumulation of these cells in the blood. It has been associated with chromosome anomaly called the Philadelphia chromosome. It is thought that 95% of patients with CML have this chromosomal abnormality. The Philadelphia chromosome originates from a reciprocal exchange of material between chromosome 22 and chromosome 9, i.e. by a translocation of genetic material. The BCR gene (breakpoint cluster region) is one of the two genes from which the BCR-ABL fusion gene and the Philadelphia chromosome are derived.

In the translocation, part of the BCR ("breakpoint cluster region") gene from chromosome 22 (region ql l) is fused with the ABLl gene on chromosome 9 (region q34) to create a mutant fusion gene BCR-ABL located on chromosome 22. Employing the International System for Human Cytogenetic Nomenclature the translocation that forms the

Philadelphia chromosome can be described as t(9;22)(q34;ql l). Nevertheless, the exact break point of chromosome 9 and 22 inside the BCR and ABLl gene is unique to the individual.

The resulting BCR-ABL fusion gene encodes for a tyrosine kinase (fusion protein also known as the Bcr-abl fusion protein). The molecular weight of the fusion protein expressed may be in the range 210 down to about 185 kDa. In CML this fusion protein usually is about

210 kDa, whereas in acute lymphocytic/lymphoblastic leukaemia (ALL) a translocation in the same region leads to a 185 kDa protein. This mutant fusion gene does not require activation by other cellular messaging proteins. In turn, the mutant fusion gene activates a cascade of proteins which control the cell cycle, speeding up cell division. Moreover, protein produced by the mutant fusion gene inhibits DNA repair, causing genomic instability and making the cell more susceptible to developing further genetic abnormalities.

This results in chronic myeloid leukaemia for which three phases have been identified namely:

• the chronic phase (the first stage), • the accelerated phase (the second stage), and

• the blast phase/crisis (the third and final stage).

In the chronic phase patients may be asymptomatic or have mild symptoms such as tiredness (fatigue), loss of appetite, weight loss, increased sweating, abnormal bruising and bleeding, a feeling of fullness or a tender lump on the left side of the abdomen, due to an enlarged spleen (splenomegaly). Swelling of the spleen may also cause pressure on the stomach, which can lead to digestive problems and poor appetite.

In the accelerated phase there may be no more symptoms than in the chronic phase, however, the number of healthy blood cells in the blood may be lower and/or the number of leukaemic cells may be higher.

The WHO criteria are perhaps most widely used and define the accelerated phase by any of the following:

• 10-19% myeloblasts in the blood or bone marrow;

• >20% basophils in the blood or bone marrow; • Platelet count <100,000, unrelated to therapy;

• Platelet count >1 ,000,000, unresponsive to therapy;

• Cytogenetic evolution with new abnormalities in addition to the Philadelphia chromosome;

• Increasing splenomegaly or white blood cell count, unresponsive to therapy.

The accelerated phase is significant because it signals that the disease is progressing toward the blast crisis (blast phase). Although this may not cause any noticeable symptoms, some people may develop high temperatures (fever) and night sweats.

In the blast phase the symptoms are likely to be more pronounced because of the increased number of abnormal white blood cells in the bone marrow and the low number of normal blood cells. The blast phase may be characterised by:

• >20% myeloblasts or lymphoblasts in the blood or bone marrow;

• Large clusters of blasts in the bone marrow on biopsy; and/or • Development of a chloroma (solid focus of leukaemia outside the bone marrow).

Symptoms include:

• having various infections one after another, caused by a lack of healthy white blood cells;

• looking pale, due to a lack of red blood cells (anaemia); • unusual bleeding, caused by a low number of platelets in the blood. This may include bruising (bruises appear without apparent injury), heavy periods in women, bleeding gums, and frequent nose bleeds. Some people may notice a particular type of bruising that consists of small blood-like spots, usually seen on the legs or in the mouth. This is called petechiae; • swollen lymph nodes;

• small nodules in the skin, caused by the spread of leukaemia cells; and/or

• generalised itching.

Chronic myeloid leukaemia has an annual incidence of 1 to 1.5 per 100,000 of the population in Italy with about 800 new case being identified each year. In the United Kingdom about 700 new cases are identified each year. It is rare in children under 10 years of age as the onset is typically between 45 and 55 years of age.

Cytogenetics is relevant to the diagnosis, typically employing chromosomal analysis of bone marrow or blood samples via fluorescent in situ hybridisation (FISH). FISH employs fluorescent probes to bind to specific targets in the BCR-ABL region of chromosome 9 and 22. The fluorescent probes bind only those parts of the chromosome with a high degree of sequence similarity thereto. The labelled chromosomes are viewed under a microscope and the translocation identified. Usually multiple rounds of fluorescence in situ hybridisation are required to identify the position of the translocation. Thus FISH mapping is time-consuming and has limited resolution.

Even after the position of the translocation has been identified for a given patient regular monitoring of diseases generated by a genomic translocation, in particular chronic myeloid leukaemia, is required. For example clinicians may want to establish if the patient is in remission and/or responding to treatment. Cytogenetics, such as FISH, can be used to count cells with the chromosomal abnormality but this is labour intensive for monitoring and may be inaccurate because only relatively small numbers of cells can be assessed, typically 200 to 500 cells are analysed, which may not be representative of the larger population of cells, given that there may be in the range 75-100 trillion cells in the human body. Thus the sensitivity of traditional cytogenetics is generally poor. For this reason those in the field of clinical research have generally moved to monitoring the mRNA products of the BCR-ABL fusion gene using techniques such as PCR. This is because reverse transcriptase PCR (RT-PCR) is a very sensitive, well established technique that can be performed on a large number of cells (such as peripheral blood cells) to provide a high level of statistical confidence in the result obtained. A number of PCR kits are available that measure the mRNA products of the BCR-ABL fusion gene. mRNA only contains transcribed exons from the genes, having already had the intronic material removed during RNA splicing. This means that information about the patient-specific break point has been removed. This permits the use of generic BCR and ABLl primers during PCR but has inherent disadvantages over measuring genomic DNA directly. Thus at the present time indirect monitoring of the behaviour of the chromosomal anomaly is performed by monitoring the mRNA expressed therefrom. There is a basic assumption that the amount of mRNA product correlates with the number of cells containing the mutant fusion gene. However, the present inventor has shown that this assumption is not necessarily correct as mRNA can have a high level of expression or a low level of expression and in some instances at the time of analysis may not be expressed at all.

Thus it now seems that the gene product generated by the abnormal cells varies between patients (and potentially between cells in a given patient) so much that it is difficult to gain meaningful insights into the disease employing only methods for analysing the presence and/or quantities of gene product present in a patient derived sample. What is more the present inventor believes that the absence, in a patient derived sample, of mRNA from the fusion gene does not in fact mean that the person/patient from which the sample was derived is free of a disease caused by a genomic translocation.

Whilst not wishing to be bound by theory the inventor believes that at certain stages of the disease leukaemic cells (i.e. cells containing a genomic translocation) may be present but are not in fact expressing mRNA product.

The inventor has some evidence which seems to show that the number of leukaemic cells does not correlate with the number expression of mRNA and this is provided in Table 2. In this table it can be seen that for patient ID 1 the levels of mRNA observed at month 0 are high whereas the levels of leukaemic cells observed are in fact relatively low. Interestingly, even though the levels of mRNA in this patient were high the patient seemed to respond to treatment very well. Thus it may be that in fact the levels of mRNA expression are not the most appropriate tool for monitoring the progression of diseases associated with a genomic translocation.

Chronic myeloid leukaemia is caused by the Philadelphia chromosome in 95% of cases (designated Ph-positive) and ultimately the presence of the chromosome is used to diagnose the disease. However, there is controversy over the existence of so called Ph-negative CML, wherein the Philadelphia chromosome cannot be detected. This inability to detect the chromosome may be due to the small sample size used in FISH or due to the level of mRNA expression being too low to be detected by RT-PCR. Thus whilst reverse transcriptase PCR (RT-PCR) is a very sensitive, well established technique that can be performed on a high number of cells (such as peripheral blood cells) to provided statistical confidence, at present, it is not used to directly measure the number of cells containing the Philadelphia chromosome (herein referred to as leukaemic cells). Rather it only detects the product of these cells, which is an indirect measurement. Although CML is perhaps the best known and understood disease generated by a genomic break point/translocation, similar problems are faced in other diseases.

Thus there is provided a method for monitoring a disease generated in a patient by a genomic translocation or providing a prognosis in relation to the disease comprising the step of: analysing an in vitro sample of at least 2,000 cells derived from said patient for the presence of a break point in genomic DNA in the sample cells, and/or the quantity of cells with a genomic break point, wherein the method employs direct one-step analysis of DNA from said sample cells.

Disease generated by a genomic translocation as employed herein is intended to refer to a disease generated by the translocation, disease derived from the translocation or disease in some way closely associated with the existence and/or creation of the translocation. Examples of diseases associated with a genomic translocation include: inflammatory myofibroblastic tumor; myelodysplastic syndrome; epithelioid hemangioendothelioma; myeloproliferative disorder, for example leukaemia such as ALL in particular T-cell acute lymphoblastic leukaemia, pre-B-cell acute lymphoblastic leukaemia, AML (acute myeloid leukaemia), chronic lymphocytic leukaemia CLL, multiple myeloma with eosinophilia, chronic myelomonocytic leukemia with eosinophilia; multiple myeloma; myxoid liposarcoma; lipoma; alveolar rhabdomyosarcoma; alveolar soft parts sarcoma; synovial sarcoma; Ewing's sarcoma; mantle cell lymphoma; hyaline vascular Castleman's disease, bizarre parosteal osteochondromatous proliferation; follicular lymphoma; follicular adenoma; anaplastic large cell lymphoma such as T/NK subtypes including diffuse large cell lymphomas; Burkitt's lymphoma; REL and diffuse large cell lymphoma, nodular lymphocyte predominant Hodgkin's lymphoma; follicular thyroid carcinoma; fibrous hamartomas; giant cell fibrosarcoma; dermatofϊbrosarcoma protuberans; sclerosing perineurioma; fibroma of tendon sheath; desmoplastic fibroblastoma/collagenous fibroma; desmoplastic small round cell tumor; pleomorphic adenoma of salivary gland, lipoma; pulmonary chondroid hamartoma; soft tissue chondroma, adenoid cystic carcinoma; renal neoplasm of children and young adults; gastrointestinal stromal tumors; low grade fibromyxoid sarcoma; endometrial stromal sarcoma; hyalinizing spindle cell tumor with giant rosettes; myxoid chondrosarcoma such as extraskeletal myxoid chrondrosarcoma; germ cell tumors; ovarian granulosa cell tumor; smooth muscle tumors (benign and malignant); pulmonary chondroid hamartomas; infantile (congenital) fibrosarcoma; cellular mesoblastic nephroma; secretory carcinoma of breast; mesenchymal chondrosarcoma; aneurysmal bone cysts including soft tissue and osseous aneurysmal bone cyst; renal cell carcinoma including pediatric renal carcinoma; and tumors.

The inventor believes that it is critical to measure the levels of abnormal cells directly, to monitor the minimal residual disease (MRD). The latter is herein defined as the small number of leukaemic cells, or cells containing a genomic break point, that remain in the patient during treatment, or after treatment when the patient is in remission.

Herein direct measurement and directly measure refer to the ability to identify the presence of a cell or cells containing a Philadelphia chromosome/fusion gene/genomic break point in a large number of cells and/or the number (quantity, percentage etc) of cells containing the translocation by a technique applied to DNA. Direct measurement is NOT indirect measurement. The latter as employed herein refers to measuring the product of DNA, be it mRNA or protein.

A large number of cells as employed herein is intended to refer to at least 2,000 cells, for example 5,000 cells, 10,000 cells, 100,000 cells, 1 million, 1.5 million, 2 million, 2.5 million, 3 million, 3.5 million, 4 million, 4.5 million, 5 million, 5.5 million, 6 million, 6.5 million, 7 million, 7.5 million, 8 million, 8.5 million, 9 million, 9.5 million, 10 million cells or more.

Monitoring as employed herein refers to analysis of patients after they have been diagnosed with a relevant disease. Prognosis as employed herein is intended to refer to predicting the patient's response or future development/progression of the disease, for example after treatment.

The method herein is NOT just the identification of the presence or absence of a breakpoint. Nevertheless the method according to the present invention can be adapted to provide a method of diagnosis with a high sensitivity and thus may be able to identify patients with an earlier stage of disease that are more suitable for treatment.

In one embodiment 5, 000 cells, 10,000 cells, 100,000 cells, 1 million, 1.5 million, 2 million, 2.5 million, 3 million, 3.5 million, 4 million, 4.5 million, 5 million, 5.5 million, 6 million, 6.5 million, 7 million, 7.5 million, 8 million, 8.5 million, 9 million, 9.5 million, 10 million, 15 million cells or more derived from said patient are analysed for the presence of a genomic break point therein.

One-step as employed herein is intended to refer to one discrete analysis without the requirement of human intervention, for example after addition of reagents the results would generally be available from the output of the instrument performing the analysis. Multiple FISH analysis to increase the representative sample size is not intended to be a one-step analysis within the meaning of the present specification. Washing of CGH slides after the hybridization stage and/or prior to scanning is not intended to be a one-step analysis within the meaning of the present specification.

Microarray/CGH/chip technology provides an indication of whether ANY of the original cells in a sample had a genomic break point, not HOW MANY had the break point.

While this could be used as a diagnostic method, it is only capable of giving a yes/no answer and thus is not a quantitative method. Furthermore, the hybridization process is a competitive process such that not all of the sample is represented in the results. Thus one cannot be certain that a negative result is not a false negative. Additionally, the technology could not be described as one-step in the context of the present specification. In one embodiment the invention does not encompass CGH technology other than its use as a means of identifying the location of the specific break point.

In one embodiment the method is qualitative in that it is able to analyse a large number of cells for the presence or absence of a genomic translocation, such as a leukaemic cell. In one embodiment the method is quantitative.

In one embodiment the method employs PCR on genomic DNA, in particular quantitative PCR.

Advantageously, DNA in PCR is less prone to degradation than RNA. In one embodiment the method employs patient specific probes and/or primers, for example based on a patient's introns.

The method according to the present invention allows large numbers of cells to analysed for any one sample, for example 500, 1,000, 500,000, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15 million cells or more can be analysed from a given sample. This makes the result of the analysis of the population of cells highly representative of the true number of cells containing a genomic translocation present in the patient. It is believed that if 10 million cells are analysed then one, for example leukaemic, cell in the population can be identified/quantified. Thus the method is also sensitive.

In one embodiment the method according the disclosure is able to identify 1 leukaemic cell in 10,000, for example 1 leukaemic cell in 100,000, 1 million, 1.5 million, 2 million, 2.5 million, 3 million, 3.5 million, 4 million, 4.5 million, 5 million, 5.5 million, 6 million, 6.5 million, 7 million, 7.5 million, 8 million, 8.5 million, 9 million, 9.5 million or 10 million cells.

The method employs an accurate/precise method of quantifying cells containing a genomic translocation, for example leukaemic cells.

Accuracy as employed herein is intended to refer to the fact the number of false positive results and/or false negative results for a given sample is low, for example 1% or less.

Precision as employed herein is intended to refer the fact the methods according to the invention provide reproducible results, provided the conditions under which the experiments/analysis is performed are controlled (for example temperature etc). Clearly the type reagents and detectors employed will influence this parameter. In contrast employing cytogenetics, at most, less than 1,000 cells could be analysed from a sample. Furthermore, FISH is considered to have an error rate of 5-10%. Thus cytogenetics is not an accurate or sensitive method of quantifying the leukaemic cells or other cells containing a genomic translocation, in the context of the present specification.

Using current methods it is difficult to detect remission in patients. FISH cannot test a representative cell sample and RT-PCR suffers from the problems described above.

The inventor has performed a comparison of quantitative analysis using the two techniques (direct and indirect techniques) on patients with chronic myeloid leukaemia undergoing treatment with the approved medication imatinib and propose the following observations from the data: • the amount of gene products expressed from leukaemic cells varies (thus there is not a linear relation between the number of leukaemic cells and the amount of mRNA produced). For example, in Table 2 when 35% of cells were shown to be leukaemic using direct (DNA) measurement (patient 1), the amount of BCR- ABL/ ABL mRNA was 228%. By contrast, in patient 10, when 93% of cells were leukaemic measuring DNA, the amount of BCR-ABL/ABL mRNA was 60%; • the absence of mRNA product does not equate to eradication of the disease (i.e. it cannot be taken as a definitive conclusion that there are no leukaemic cells because it may be that immature or pre-cursor leukemic cells can be detected directly which are not expressing mRNA but have the potential to do so); and • that the absence of leukaemic cells may be a good indicator that the patient is in remission.

Whilst it has been hypothesised that imatinib (and other treatments such as dasatinib and nilotinib) may provide long term remission for some patients, physicians have been reluctant to stop treatment because many patients simply relapse. Thus whilst it may be that as many as

20% of patients could discontinue treatment, it is continued because those who would benefit from discontinuing cannot be identified based on current methods.

This is a real problem for patients and health authorities. Firstly from the patients perspective imatinib is effective and has improved treatment for chronic myeloid leukaemia immensely. However, it has a number of side effects including: nausea; vomiting; swelling in the face, especially around the eyes; diarrhoea; leg aches and cramps; an itchy rash; poor appetite; headaches; and tiredness. Clearly if the medication is not required it would be better to avoid these side-effects.

Secondly the cost of the medication is about £28,032.00 (GBP) per patient per year. Continuing treatment for patients who no longer require it is not a cost effective way of spending the limited money available to health authorities. Therefore, it is in the patients and health authorities interests to robustly be able to identify those patients in remission for whom treatment is no longer required.

Thus a reliable method of monitoring patients with a disease associated with a genomic translocation would be useful to clinicians, in particular for predicting the prognosis of the patients and indentifying those who may be in remission.

A patient may be considered to be in remission if the result of the analysis is that: there are no cells identified with a genomic translocation, or the number of cells with a genomic translocation is constant or lower on one or more occasions (such as the result of 3 consecutive analysis over a 1 to 6 month period) than the results of a previous or initial analysis.

The method of the present invention may also be suitable for identifying those patients with the potential to respond well to therapy, for example those patients with a low percentage of cells with a translocation regardless of the amount of product expressed by those cells. Thus there is provided a method of identifying a chronic myeloid leukaemia patient in remission or not in remission comprising the step of quantitative assaying the amount of leukaemic cells in a sample derived from a patient, for example bone marrow or peripheral blood, by directly measuring the quantity of cells with a genomic break point which form a Philadelphia chromosome. The method also provides a method of monitoring a patient with myeloid leukaemia and may provide valuable information on the likely prognosis and/or progression of the patient.

In one embodiment the assay is directed at the BCR-ABL gene in the Philadelphia chromosome. The assay can employ any quantitative method for genomic DNA. However, quantitative real time PCR (Q-PCR) is a particularly suitable technique because it is sensitive, robust and is widely available in laboratories throughout the world.

Having said this, given that the translocation which forms the 9:22 break point is unique to the individual, specific primers have to be identified for each patient.

Methods for Identifying the Location of the Break Point

There are several methods that can be used to identify the location of the break point for a given patient and then design primers and/or probes based on the same for use in the method of the present invention. These methods include:

FISH;

Array painting, see for example Nature protocols 2009 VoI 4 No 12 page 1722-1736,

Comparative genomic hybridization (CGH) see for example WO 2009/062166, and

Sequencing the genome or relevant part thereof, for example using Illumina type technology. These methods will be discussed in more detail below.

Any of the above steps or alternative method for identifying the position of the break point may be employed in combination with the method of the present invention. Before the analysis to identify the break point is performed some sample preparation may be required, for example derivative chromosome isolation.

Derivative Chromosome Isolation by Flow Sorting

In order to identify the precise break point in every patient white cells from a peripheral blood sample are collected and then lysed. A method for extracting the cells from a peripheral blood sample is provide in the Examples. Chromosome sorting is performed for example on a fluorescence activated cell sorting (FACS) Vantage™ (available from BD) cell sorter equipped with two lasers that emit in the UV range, for example at 330-360 nm and 457nm respectively, with 200 mW output each. Chromosome suspensions are stained with H33258 and

Chromomycin A.

The purity of the sort is determined by extensive quality controls. Samples of sorted chromosomes are fixed on slides and hybridized with a probe specific for the sorted chromosome. Depending on the quality of the chromosome preparation a purity of more than

95% can be achieved. (Weier HU Genomic, 1994 Jun;21(3):641-4).

The DNA derived from the two chromosomes is then digested and used separately to hybridize a comparative genomics hybridization (CGH) slide. The two hybridizations will indicate in each patient the sequences present on chromosome 22 flanking the sequence on chromosome 9. The Philadelphia chromosome and its derivative are digested and hybridized to a slide such as a CGH slide, which enables the identification of the position of the break point. A more efficient method is to employ microchips designed specifically to cover only the genomic region of interest. The advantage of this customized DNA chip apart from the price is that the spots will represent sequences laying 800 bp or less from each other. The primers and/or the probes can be designed directly in the sequence across the break point from chromosome 9 to 22.

There is provided in one embodiment a kit comprising a microarray chip designed to cover the chromosomes comprising a breakpoint, for example 9 and/or 22 translocation regions involved in forming a Philadelphia chromosome wherein the oligos laid down on the chip are up to 60 nucleotides long and the sequence coverage is no more than 160 base pairs apart and reagents and/or instructions for use in the analysis according to the present invention.

The length of the oligos is designed according to the target sequences. Generally the longer the oligos on the chip the more specific to the site they will be. Each oligo may be of the length 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28,

29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 or 60 nucleotides.

As mentioned above the primers and the probe are designed across the genomic break point of each patient. The reason they are specific to each patient is because the chromosome breakage can occur in any region of the introns (non-coding regions) of, for example BCR and

ABLl, which vary in each individual. Thus in one embodiment the method according the disclosure herein includes the step of identifying where the translocation is situated for a given patient and optionally preparing primers to bind specifically in said location.

Once designed the primers and probe will remain relevant to a given patient, for as long as the disease is present and can then be used to monitor the number of cells (for example

Ieukaemic cells) during the patient's therapy. This is particularly true when the technique used to monitor the cells is PCR.

The length of the primers/probes for PCR is designed according to the target sequences.

Generally the longer the primers/probes the more specific to the site they will be. Each primer may be of the length 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides, in particular 15, 16, 17, 18, 19 or 20, most particularly 18, 19, 20, 21, 22, 23 or 24. Examples of certain patient specific primers are shown in Table 1, see also

WO2009062166. Obviously, it takes a certain amount of time to determine the primers/probes required for a given patient but once identified these primers/probes are suitable for monitoring throughout the patient's lifetime. Therefore, it would be advisable to record the position of the translocation and/or suitable primers/probes on the patient's medical records, to avoid the need to repeat this work. Additionally, given there are a limited number of patients with chronic myeloid leukaemia and given:

• the treatment is very expensive;

• unnecessary side-effects for individuals should be avoided; and

• the cost of the assays is likely to be moderate, it makes sense to invest the time to provide this individualised care to the patients.

Once the patient specific primers are identified then PCR can be performed in the usual way to identify and/or quantify the cells with the translocation.

In one embodiment the method may in addition contain the step of performing a PCR assay based on mRNA, for example RT-PCR. Of course it may be useful to have a comparison of the "Ieukaemic cell assay" with a quantitative assay for gene products. For example, where there are only a low number of cells with the genomic translocation, it may be of interest to test how much mRNA or protein is being produced by those cells to gain further insights into the disease. PCR analysis

Suitable kits include: BCR/ABL1 Quant (RUO) kit Asuragen Inc. a research tool utilizing multiplex real-time quantitative RT-PCR to provide simultaneous detection and quantification of BCR/ABLl fusion transcripts (b2a2, b3a2, and ela2), ABLl (an endogenous control), and BCR/ABLl Quant Norm (an exogenous control) in a single reaction.

The kit is based on TaqMan(R) technology and is compatible with ABI 7500 real-time systems or equivalent. Fusion transcripts corresponding to BCR/ABLl b2a2 or b3a2 are present in >95% of CML patients. About 5% of children with acute lymphoblastic leukaemia (ALL) and 20-35% of adult ALL also carry t(9;22), most often ela2. This molecular assay provides sensitive quantitation of the BCR/ABLl transcript from RNA extracted from peripheral blood, bone marrow aspirates or cultured cells. BCR/ABLl Quant is an assay providing broad target coverage and dynamic range with internal and external assay calibration powered by Asuragen's Armored RNA(R) Quant(TM) (ARQ) Technology. The kit includes an optional exogenous internal spike in ARQ control, BCR/ABLl Quant Norm, to assess process efficiency and 4 external calibrators consisting of a blend of precisely quantified BCR/ABLl, ABLl and BCR/ABLl Quant Norm ARQs mixed at different concentrations to generate 3 standard curves. The resulting PCR products are compatible with capillary electrophoresis (CE) for subsequent determination of the fusion transcripts identity (ela2, b2a2, or b3a2) via size fractionation. The assay of the present disclosure can easily be performed on a sample derived from peripheral blood having the advantage of a less invasive sampling. Alternatively, a sample can be derived from a bone marrow biopsy from a patient.

Blood samples can be taken daily, weekly, monthly, for example every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, as required. Bone marrow samples, realistically, can only be taken once a year because of the invasive nature of the testing.

The analysis of bone marrow samples may provide additional information in that it allows the analysis of the presence of abnormal stems cell, i.e. undifferentiated cells, which are primarily located in the bone marrow. Zero abnormal stem cells provides a high level of confidence that the patient is in remission.

Analysis of a (one) patient derived sample may be performed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times, thereby allowing a larger number of cells to be analysed. Routinely, the analysis may be performed 2, 3 or 4 times on a sample.

The method optionally includes the step of analysing the results and assigning the patient to the category of in remission or not in remission, for example as defined herein above. In one embodiment the method comprises the further step of stopping/suspending or reducing treatment for a disease associated with a genomic break point for a patient identified as remission or with mild residual disease.

The sensitivity of the genomic assay revealed the persistence of leukaemic cells at 0.001% (Table 2). Patients with levels of leukemic cells above this may be considered not to be in remission and therefore in need of further treatment. Patients in remission may generally have undetectable level of leukemic cells.

Thus in one embodiment there is provided a method of treating a patient with chronic myeloid leukaemia wherein the patient is identified as in remission by a method herein comprising the step of stopping imatinib treatment (or corresponding treatment such as dasatinib or nilotinib) and optionally monitoring the patient for presence and/or the quantity of leukaemic cells.

The monitoring could be as frequent as the physician may decide because the samples analysed can be peripheral blood and thus it is not necessary to employ bone marrow samples. It may, for example be daily, weekly, monthly, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 monthly, such as 1 monthly initially but then may be moved to 3 monthly or 6 monthly. If the physician is confident that the remission has been sustained for a long period then the monitoring may be annually.

Clearly one or more steps of the assay herein may be repeated for each time there is a retest.

If the patient becomes immunosuppressed, for any reason, such as pregnancy, then monitoring should be increased or maintained at short intervals because relapse can occur in these circumstances.

In one embodiment there is provided a kit comprising the components for an assay according to the invention and/or instructions on how to perform the assay.

In a further aspect there is provided a method of diagnosing chronic myeloid leukaemia comprising the step of quantitatively assaying the amount of leukaemic cells in a patient from a sample, derived from bone marrow or peripheral blood, by directly measuring the leukaemic cells, analysing the results thereof and assigning the patient to the category of chronic myeloid leukaemia or non-chronic myeloid leukaemia.

The results shown in Table 2 seem to indicate that in the vast majority of cases the % of leukaemic cells in a patient with chronic myeloid leukaemia, for example at the exordium, is high such as 60, 65, 75, 80% or more. This data, for example obtained at the exordium, could have a clinical value to evaluate the possible prognosis of every single patient. This numeric value also seems to be a reliable way of identifying those with the disease. The method described above for identifying if a patient is in remission can be adapted to provide a method of diagnosis employing the methods described herein.

In a further aspect there is provided a method of predicting the prognosis for a patient already identified as a chronic myeloid leukaemia sufferer comprising the step quantitative assaying the amount of leukemic cells in a patient from a sample, derived from bone marrow or peripheral blood, by directly measuring the cells with a break point which form a Philadelphia chromosome, analysing the results thereof and assigning a patient as likely to progress into remission following treatment, for example with imatinib, or as a patient likely to require long term maintenance treatment. The results in Table 2 for patient ID No: 1 may indicate that a low level of leukaemic cells, for example even in the presence of high levels of mRNA may render the patient particularly conducive to treatment, for example with imatinib, and therefore likely to go into remission.

The method of the present invention can also be used as a robust tool to monitor the effectiveness of new and/or experimental treatments for cancers such as chronic myeloid leukaemia, for example in clinical trials.

It is also believed that there may be a correlation with the quantity of cells with the translocation and the dose of medicament required for treatment, such as the dose of imatinib.

Thus when the patient is not in remission, of chronic myeloid leukaemia, but has only low levels of leukaemic cells then a lower dose for example 200-400mg/day of drug may be required for effective treatment or maintenance of the disease. If there are larger numbers of leukaemic cells then higher doses such as 600-800mg/day may be required. The present method provides the opportunity to give the patient the most appropriate dose for their specific symptoms. Thus there is provided a method of treating a patient comprising the step of assessing accurately the number of cells with a translocation and then administering a dose of medicament, for example imatinib selected specifically for the patient based on the number of leukaemic cells identified.

Thus there is provided herein a tool for disease management and/or control and monitoring.

The methods employed above for chronic myeloid leukaemia may also be employed to identify and monitor translocations in other tumours, cancers and diseases that result in hyper- functionality or gain of function of a new gene. Examples of other translations locations are:

Disease Associated with Translocations in chromosome 1

Mucosa-associated lymphoid tissue (MALT) lymphoma is sometimes associated with a translocation t(l;2)(p22;pl2): related proteins are BCLlO and kappa light chain and/or translocation t(l;14)(p22;q32): related proteins BCLlO and IgH;;

Inflammatory myofibroblastic tumor is associated with translocation t(l;2)(q22- 25;p23): related proteins tropomyosin alpha-3 chain and ALK. The translocation is rare in anaplastic large cell lymphoma;

Myelodysplastic syndrome is sometimes associated with translocation t(l;3)(p36;q21): related proteins are MELl and RPNl and/or translocation t(l;7)(qlθ;plθ): proteins unknown; Epithelioid hemangioendothelioma is associated with translocation t(l;3)(_P36.3;q25);

T-cell Acute lymphoblastic leukaemia is associated with translocation t(l;7)(p34;q34): related proteins LCK and TCR beta;

Up to 30% of pre-T-cell Acute lymphoblastic leukaemia patients have a translocation t(l;14)(p32;qll): related proteins TAL1/SCL and T cell receptor alpha/delta;

Alveolar rhabdomyosarcoma can be associated with a translocation t(l;13)(p36;ql4): related proteins PAX7 and FKHR; and/or the translocation t(2;13); mantle cell lymphoma and pre-B-cell Acute lymphoblastic leukaemia may be associated with translocation t(l;14)(q21;q32): related protein BCL9 and IgH; pre-B-cell Acute lymphoblastic leukaemia may be associated with translocation t(l;14)(q25;q32): related proteins LHX4 and IgH and/or translocation t(l ;19)(q23;pl3.3): PBXl and E2A:- references: MoI Cell Biol 1994;14:3938; hyaline vascular Castleman's disease may be associated with translocation t(l;16)(pll;pll):- references: AJSP 2000:24:882 bizarre parosteal osteochondromatous proliferation may be associated with translocation t(l;17)(q32;q21):- reference Hum Path 2004:35:1063: AML-M7 in infants may be associated with t(l;22)(pl3;ql3): related proteinsOTT and MAL:- references: Genes Chromosomes Cancer 2002:33:22, Blood 2002;100:618;

Ewing's sarcoma/PNET may be associated with translocation t(l;22)(p36.1;ql2): ZSG and EWS genes:- references: Oncogene 2000:19:3799 follicular lymphoma may be associated with the translocation t(l;22)(q22;qll): FC gamma RIIb and lambda light chain:- references: OMIM 604590

Disease Associated with Translocations in chromosome 2 Anaplastic large cell lymphoma may be associated with translocation t(2;3)(p23;q21): ALK and TFG (tropomyosin receptor kinase fused gene); follicular thyroid carcinoma and/or follicular adenoma may be associated with translocation t(2;3)(ql3;p25): related proteins PAX8 and PPAR gamma 1 :- references: AJSP 2002:26: 1016, Am J Path 2005:167:223; fibrous hamartomas may be associated with translocation t(2;3)(q31;q21): unknown proteins; reference Archives 2005:129:520; anaplastic large cell lymphoma (TYNK subtypes, 40-70%) and/or inflammatory myofϊbroblastic tumor may be associated with translocation t(2;5)(p23;q35): related proteins ALK and NPM:- references: Blood 1989;73:806 (early report), Blood 1996;87:284 , Mod Path 2002;15:931, AJSP 2003:27:1473;

Burkitt's lymphoma and/or mantle cell lymphoma may be associated with translocation t(2;8)(pl2;q24): related proteins kappa light chain and c-myc:- reference Mod Path 2002; 15: 1266 (mantle cell lymphoma); REL and diffuse large cell lymphoma may be associated with translocation t(2,8)(pl2-16;q24):- references: Blood 2004:103:1862 sclerosing perineurioma may be associated with translocation t(2;10)(p23;q24): proteins unknown:- reference AJSP 2005:29:1164); fibroma of tendon sheathand/or and desmoplastic fibroblastoma/collagenous fibroma may be associated with translocation t(2;ll)(q31-32;ql2):- references Histopathology 1998:32:433, Cancer Genet Cvtogenet 2004:149: 161 ; alveolar rhabdomyosarcoma may be associated with transloction t(2;13)(q35;ql4): related proteins PAX3 and FKHR:- references: Genes Chromosomes Cancer 1995:12:186, Am J Path 1995:146:626. AJSP 2002:26:938; CLL and/or ALL and/or AML may be associated with translocation t(2;14)(pl3;q32): BCLl IA and IgH;- references: Leukemia 2002:16:937. Blood 2001:98:3413 (free). Leuk Lymphoma 2002:43:2063 (case report); inflammatory myofibroblast^ tumor and/or anaplastic large cell lymphoma may be associated with translocation t(2;17)(p23;q23): related proteins ALK and CLTC:- references: Am J Path 2001 :159:411 (free).-Mod Path 2003:16:828: follicular lymphoma and/or CLL/SLL may be associated with translocation t(2;18)(pll-12;q21): related proteins kappa light chain and BCL2:- references: Leuk Lymphoma 1992:8:197. Oncogene 1992:7:573, Br J Haematol 1991 :78:132; inflammatory myofibroblastic tumor may be associated with translocation t(2;19)(p23;pl3.1): ALK and TPM4:- references: Am J Path 2000;157:377; Ewing's sarcoma/PNET may be associated with translocation t(2;22)(q33;ql2): FEV and EWS:- references: Oncogene 1997:14:1159

Disease Associated with Translocations in chromosome 3 Myelodysplastic syndrome, acute myeloid leukemia with multilineage dysplasia may be associated with translocation t(3;5)(q25;q34-35): MLFl and NPM:- references: Hum Path 2003:34:809 (AML and myelodysplasia), Leukemia 2000:14:1757: pleomorphic adenoma of salivary gland may be associated with translocation t(3;8)(p21;ql2): related proteins CTNNBl and PLAGl :- references: Mod Path 2005:18:1048, Nat Genet 1997:15:170: lipoma, pulmonary chondroid hamartoma and/or soft tissue chondroma may be associated with translocation t(3;12)(q27;ql4-15): related proteins HMGA2 and LPP:- references Genes Chromosomes Cancer 1998:22:100. Mod Path 2003:16:1132 (chondroma); diffuse large B cell lymphoma and/or follicular lymphoma and/or nodular lymphocyte predominant Hodgkin's lymphoma may be associated with translocation t(3;14)(q27;q32): BCL6 and IgH: reference J MoI Diagn 2005:7:352;

CML and/or myelodysplastic syndrome may be associated with translocation t(3;21)(q26;q22): EVIl and AMLl :- references: Oncogene 2004:23:4263;

Disease Associated with Translocations in chromosome 4 preB ALL and/or post-treatment ALL and/or AML M4/M5 may be associated with translocation t(4;ll)(q21;q23): related protein AF4 and ALL1/MLL:- reference Ann Hematol 1992:65:143, Blood 2005:105:3434; multiple myeloma may be associated with translocation t(4;14)(pl6;q32): related protein FGFR3 and IgH:- references: Blood 2005; 105:4060. Clin Cancer Res 2004:10:5692;

Disease Associated with Translocations in chromosome 5

ALL with eosinophilia may be associated with translocation t(5;9)(q31;p24): related proteins IL3 and JAK2 genes:- references Archives 2003;127:601 chronic myelomonocytic leukemia with eosinophilia may be associated with translocation t(5;12)(q33;pl3): PDGFRB and ETV6:- references: Acta Haematol

2002:107:113: preB ALL with peripheral eosinophilia may be associated with translocation t(5;14)(q31 ;q32): related protein IL3 and IgH;

Disease Associated with Translocations in chromosome 6 AML and/or myelodysplastic syndrome may be associated with translocation t(6;9)(p23;q34): DEK and CAN:- references: Leukemia 2005:19:1338, AJCP 1997:107:430. Blood 1992;79:2990; adenoid cystic carcinoma may be associated with translocation t(6;9)(q21- 25;pl3-24):- references: Eur J Oral Sci 2004:112:545. Genes Chromosomes Cancer 2001:30:161 : renal neoplasm of children and young adults may be associated with translocation t(6;ll)(p21;ql2-13); related proteins TFEB and Alpha: references: AJSP 2005:29:230. Proc Natl Acad Sci USA 2003; 100:6051, Hum MoI Genet 2003:12: 1661. OMIM 600744 hyaline vascular Castleman's disease may be associated with translocation t(6;12)(q23;ql5): related proteins HMGA2/HMGIC:- references: AJSP

2002:26:662 gastrointestinal stromal tumors and/or, multiple myeloma and/or diffuse large B cell lymphoma may be associated with translocation t(6;14)(p21.1 ;q32.3); related proteins cyclin D3 and IgH:- references: Mod Path 2003:16:886 TGIST): multiple myeloma may be associated with translocation t(6;14)(p25;q32): related protein MUM/IRF4 and IgH:- references: Leukemia 1999; 13:1812

Disease Associated with Translocations in chromosome 7

T-cell ALL may be associated with translocation t(7;9)(q34;q34.3): TCR beta and TAN1/NOTCH1:- references: Cell 1991 :66:649. Cancer Lett 2005:219:113; low grade fibromyxoid sarcoma, hyalinizing spindle cell tumor with giant rosettes t(7;16)(q34;pll): proteins unknown:- references AJSP 2003:27:1229. Archives 2000; 124: 1179; endometrial stromal sarcoma may be associated with translocation t(7;17)(pl5;q21): related proteins JAZFl and JJAZl:- references: AJSP 2004:28:224. Proc Natl Acad Sci USA 2001:98:6348 (free). Cancer Genet Cvtogenet 2003:144:119. J MoI Diagn 2005:7:388. OMIM 606246 T-cell ALL may be associated with translocation t(7;19)(q34-35;pl3): related proteins TCR beta and LYLl : reference MoI Cell Biol 1996:16:2394). Ewing's sarcoma/PNET may be associated with translocation t(7;22)(p22;ql2): related proteins ETVl and EWS:- references OMIM 600541 -ETVl. Cancer Res 2000:60:1536

Disease Associated with Translocations in chromosome 8

B-cell ALL may be associated with translocation t(8;9)(q24;pl3): related protein c-myc Archives 2003;127:610-ease report) myeloproliferative disorder s such as T cell lymphoblastic lymphoma may be associated with translocation t(8;13)(pll-12;qll-12): related proteins FGFRl and ZNF 198:- references: Nat Genet 1998:18:84. Acta Haematol 2002:107:101; Burkitt's lymphoma and/or ALL-L3 and/or mantle cell lymphoma may be associated with translocation t(8;14)(q24;q32.3): related proteins c-myc and IgH:- references AJSP 2003:27:818 (Burkitt's in transplant recipients). Mod Path 2002:15:1266 (mantle cell lymphoma). Leukemia 2003:17:585; AML-M2 with Auer rods and/or granulocytic sarcoma may be associated with translocation t(8;21)(q22;q22): related proteins ETO and AMLl :- references Nat Med 2002;8:743 (free), Proc Natl Acad Sci USA 2005;102:4016; Burkitt's lymphoma may be associated with translocation t(8;22)(q24;qll): elated proteins c-myc and lambda light chain;

Disease Associated with Translocations in chromosome 9

AML-M5a and M4 and/or therapy related AML and/or ALL may be associated with translocation t(9;ll)(p22;q23): related proteins AF9 and MLL/ ALLl :- references Genes Chromosomes Cancer 1991:3:74, Hum MoI Genet 2000;9:1671 ; lymphoplasmacytic lymphoma and/or diffuse large B cell lymphoma and other B cell lymphoproliferative disorders may be associated with translocation t(9;14)(pl3;q32): related proteins PAX5 and IgH: reference Blood 1996:88:4110. Genes Chromosomes Cancer 2005:44:218. Hum Path 2004:35:447 (not characteristic for lymphoplasmacytic lymphoma), Leuk Lymphoma 2000:36:435; extraskeletal myxoid chrondrosarcoma may be associated with translocation t(9;15)(q22;qll-q21): related proteins TEC/CHN and TCF12:- references: Am J Path 2003:162:781. Cancer Res 2000:60:6832 myxoid chondrosarcoma may be associated with translocation t(9;17)(q22;qll- 12): related protein TEC/CHN and TAF2N/RBP56; [variant of t(9;22)]:- reference Cancer Res 1999:59:5064. Am J Path 2003:162:781 (free). Hum Path 2001:32:1116. Oncogene 1999; 18:7594. AJSP 2000;24: 1020. TAF2N; extraskeletal myxoid chondrosarcoma may be associated with translocation t(9;22)(q22-31;qll-12): related proteins TEC/CHN and EWS:- references Mod Path 1995:8:765. Cytopathology 1991:2:261. Genes Chromosomes Cancer 2002:35:340 CML (100%), preB ALL (5% of children, 25% of adults), and/or AML may be associated with translocation t(9;22)(q34;qll): related proteins c-abl and bcr (Philadelphia chromosome):- references: Mayo Clin Proc 2005:80:390. Clin Lab Sci 2005:18:38. more information (CML).

Disease Associated with Translocations in chromosome 10 preT-cell ALL may be associated with translocation t(10;14)(q24;qll): related proteins HOXI l and T cell receptor delta:- references: Proc Natl Acad Sci USA 1990:87:3161 (free). Leuk Lymphoma 1995;16:209. low grade non-Hodgkin's lymphomas may be associated with translocation t(10;14)(q24;q32): related proteins NFKB-2/LYT10 and IgH;- references: CeU 1991;67:1075. OMIM 164012 (NFKB-2)

Disease Associated with Translocations in chromosome 11

AML (frequent), ALL (10% involve 1 Iq23 rearrangements, less involve self- fusion) may be associated with translocation t(l 1 ;1 1)(q23;q23): MLL/ALL1 (self-fusion):- references: Proc Natl Acad Sci USA 1998:95:2390 (free). Leuk Res 2005:29:517, Blood 1996;87:2496 (free). Cancer Res 1997:57:117: T-cell ALL (5% of childhood cases) may be associated with translocation t(ll;14)(pl3;qll): related proteins rhombotin 2 (TTg-2, RBTN2) and T cell antigen receptor alpha/delta:- references: Leukemia 1995:9:1812;

T-cell ALL (<1%) may be associated with translocation t(ll;14)(pl5;qll): related protein rhombotin 1 (TTg-I /LMOl) and TCR alpha/delta; References: MoI Cell Biol 1989;9:2124; mantle cell lymphoma (90%), B cell prolymphocytic leukemia (20%, may represent mantle cell variant) and/or splenic lymphoma with villous lymphocytes (10%) and/or CLL (2-5%) and/or myeloma (2-5%) may be associated with translocation t(l I;14)(ql3;q32): BCLl/cyclin Dl and IgH:- reference Br J Haematol 2004; 125:330). Blood 1996;88:674, Archives 1999:123:1182..Hum Path 2002:33:7; AML M3 variant may be associated with translocation t(ll;17)(q23;q21): related proteins PLZF and retinoic acid receptor-alpha:- references: Semin Hematol 2001 :38:37. Proc Natl Acad Sci USA 1997;94: 10255; MALT lymphoma (50%); also diffuse large B cell lymphoma may be associated with translocation t(ll;18)(q21;q21): related proteins API2 and MALTl:- references: Mod Path 2003; 16: 1232 (colorectal lymphomas), Int J Hematol 2005:82:59 (cytologic specimens);

AML and/or M4/M5; also M1/M2 may be associated with translocation t(ll;19)(q23;pl3): related protein ALLl and ELL:- references: Proc Natl Acad Sci USA 1994:91 : 12110, Cancer Genet Cvtogenet 2001:129:17 (case report); desmoplastic small round cell tumor may be associated with translocation t(ll;22)(pl3;ql2): related proteins WTl and EWS:- references: AJSP 2002:26:823. Archives 2002:126:1226 (lung tumor) [correction at Archives 2003:127:7821. Mod Path 2002:15:673 (dural tumor). AJSP 1992; 16:411 (original report). Semin Cancer Biol 2005;! 5: 197; Ewing sarcoma/PNET (90% of cases) may be associated with translocation t(ll;22)(q24;ql2): related proteins FLIl and EWS: references: Adv Anat Pathol 2005:12:212. Cancer Res 2005;65:4633;

Disease Associated with Translocations in chromosome 12 myelodysplastic syndrome and/or germ cell tumors may be associated with a translocation in 12q:- references: Archives 2005:129:1299, Cancer Genet

Cvtogenet l995;80:158

CLL/SLL (10-30%). ovarian granulosa cell tumor:- references Blood

1993;82:571, J Clin Oncol 1984;2: 1121, translocation in i(12p) may be associated with intratubular germ cell neoplasia; germ cell tumors:- references: Mod Path 2005:18 Suppl 2:S51. APMIS

2003:111 :161; smooth muscle tumors (benign and malignant), lipoma, pleomorphic adenoma of salivary gland and elsewhere, pulmonary chondroid hamartomas may be associated with translocation t(12;14)(ql4-15;q23-24): related proteins HMGA2/HMGIC and various:- references: Cancer Genet Cvtoeenet 1988:32:13 (uterine leiomyomas), Cancer Genet Cytogenet 2002; 138:50 (various smooth muscle tumors). Mod Path 2002:15:351 (intravenous leiomyomatosis), more information-HMG A2 ; AML and/or infantile (congenital) fibrosarcoma, cellular mesoblastic nephroma, secretory carcinoma of breast may be associated with translocation t(12;15)(pl3;q25): related proteins ETV6 and NTRK3; (Genes Chromosomes Cancer 2004:40:152), (Blood 1999:93:1355 (case report). Mod Path 2000:13:29, Mod Path 2001:14:1246, AJSP 2000:24:937. Nat Genet 1998:18:184, Pathol Res Pract 2003:199:35, Hum Path 2003:34:1299 (secretory carcinoma); myxoid and round cell liposarcoma, rarely epithelioid variant of pleomorphic liposarcoma may be associated with translocation t(12;16)(ql3;pll): related proteins CHOP and TLS:- reference Histopathology 2005:46:334. J MoI Diagn 2000;2:132, Semin Diagn Path 2001 :18:267 (review), more information-TLS. more information-CHOP/DDIT3: preB-cell ALL (20%) may be associated with translocation t(12;21)(pl2- 13;q22): related protein TEL/ETV6 and AML1/CBFA2:- references: Curr Opin Hematol 2002:9:345, Diagn MoI Path 2000:9:184; AML may be associated with translocation t(12;22)(pl3;qll-12): related proteins TEL/ETV6 and MNl :- references: MoI Cell Biol 2000:20:9281. more information, MNl -more information. ETV6-more information; myxoid liposarcoma may be associated with translocation t(12;22)(ql3;ql2): related proteins CHOP and EWS:- references: J MoI Diagn 2002:4:164, Clin Cancer Res 2000:6:2788 ), more information-CHOP/DDIT3; clear cell sarcoma of soft parts (>95%) may be associates with translocation t(12;22)(ql3;ql2): related proteins ATFl and EWS;

Disease Associated with Translocations in chromosome 13

Mesenchymal chondrosarcoma may be associated with translocation der(13;21)(qlθ;qlθ): proteins unknown: references Mod Path 2002;15:572

Disease Associated with Translocations in chromosome 14

Diffuse large cell lymphomas (4%) may be associated with translocation t(14;15)(q32;qll-13): related proteins IgH and BCL8:- references: Proc Natl Acad Sci USA 1997;94:5728; multiple myeloma (10%) may be associated with translocation t(14;16)(q32;q23): related proteins IgH and c-maf:- references: Blood

1998;91:4457 follicular lymphoma (90%); secondary but not primary cutaneous follicular lymphoma, B cell ALL; diffuse large cell lymphoma (30%, probably of follicle center origin), and/or rarely CLL may be associated with translocation t(14;18)(q32;q21): related proteins IgH and BCL2:- references: Archives 2005:129:410, Am J Path 2002:160:759, AJSP 2003;27:356, Archives 2003:127:610, Archives 2002:126:1543 (CLL), Archives 2002:126:902: MALT lymphomas (20%) may be associated with translocation t(14;18)(q32;q21): related protein IgH and MALTl :- references: Blood 2003;101 :2335;

CLL/SLL (5%) may be associated with translocation t(14;19)(q32;ql3): related protein IgH and BCL3:- references: Leuk Lymphoma 2002:43:813, Genes Chromosomes Cancer 1997:20:64

Disease Associated with Translocations in chromosome IS

AML-M3 (acute promyelocy e leukemia) may be associated with translocation t(15;17)(q22;ql2-21): related protein PML and retinoic acid receptor-alpha:- references: Acta Haematol 2004:1 12:55. Curr Oncol Rep 2003:5:391,

Disease Associated with Translocations in chromosome 16 aneurysmal bone cysts may be associated with translocation t(16;17)(q22;pl3): related protein CDHl 1 and USP6:- references: Cancer Res 2004:64:1920 ffree). Genes Chromosomes Cancer 1999:26:265: multiple myeloma may be associated with translocation t(16;22);(q23;qll): related proteins c-maf and Ig lambda:- references: Blood 1998:91 :4457

Disease Associated with Translocations in chromosome 17 soft tissue and osseous aneurysmal bone cyst may be associated with translocation t(17;17)(pl3;ql2): related protein USP6 and COLlAl :- references: AJSP 2002:26:64, Oncogene 2005:24:3419; Ewing's sarcoma/PNET (rare) may be associated with translocation t(17;22)(ql2;ql2): related protein ElAF and EWS:- references: Cancer Lett 2004:216:1, Jpn J Cancer Res 1998:89:703; dermatofibrosarcoma protuberans, giant cell fibrosarcoma may be associated with translocation t(17;22)(q21-22;ql3) or related ring chromosomes: related proteins COLlAl and PDGF beta:- references: Oncogene 2001 :20:2965. AJSP 2003;27:27, more information-PDGF beta

Disease Associated with Translocations in chromosome 18

<5% of CLL/SLL may be associated with translocation t(18;22)(q21;q21): lambda light chain (22ql l) and BCL2 (18q21):- references: Genes Chromosomes Cancer 1993;6:39, Genes Chromosomes Cancer 1991;3:205, Leukemia 1996;10:970

Disease Associated with Translocations in chromosome 20 myelodysplasia syndrome, myeloproliferative disorders and/or AML, myelomas may be associated with a translocation in region 2Oq:- reference Genes Chromosomes Cancer 2004;41 :223, Cancer Genet Cytogenet 2005:160:188; trisomy associated with desmoid-type fibromatosis and/or patients at risk for lung cancer may have a link with a translocation is region 20: Am J Path 1999:154:729. Int J Cancer 1995:63:527, Cancer Epidemiol Biomarkers Prev 1998:7:1051

Disease Associated with Translocations in chromosome 21 Ewing's sarcoma/PNET (5-10%) and/or desmoplastic small round cell tumor may be associated with translocation t(21;22)(q22;ql2): related protein ERG and EWS:- AJSP 1998:22: 1026, Nat Genet 1994:6: 146, J Clin Oncol 1999:17:1809

Translocations - chromosome 22 CML variant Philadelphia chromosome may be associated with translocation t(22;22)(ql3;ql 1): related protein bcr:- references: Leuk Res 1986:10:1131, Blut

1989:58:279

Translocations - chromosome X

Certain subtypes of renal cell carcinoma may be associated with translocation t(X;l)(pll.2;q21.2): related protein TFE3 and PRCC:- references: AJSP

2002:26:1553, anaplastic large cell lymphoma may be associated with translocation t(X;2)(qll;p23): related proteins MSN and ALK; subungual exostosis; also cases reports of AML-M7, premature ovarian failure, female with Duchenne muscular dystrophy may be associated with a translocation in region t(X;6):- references: AJSP 2004:28: 1033 pediatric renal carcinoma, alveolar soft parts sarcoma may be associated with translocation t(X;17)(pll.2;q25): related protein TFE3 and ASPL:- references:

AJSP 2003:27:750 synovial sarcoma (>90%) and some other tumors may be associated with translocation t(X;18)(pll.2;qll.2): SYT and SSXl or SSX2, rarely SSX4;

Thus one aspect the disclosure provides a method of diagnosing or monitoring a tumor, cancer or disease resulting from a hyperfunction generated by a cell with a translocation, comprising the step of directly identifying and/or quantifying the cell with translocation, wherein the method does not employ cytogenetics to identify a translocation which forms the

Philadelphia chromosome.

The methods and processes described above in relation to chronic myeloid leukaemia apply equally to alternative cancers, tumours and diseases that have a translocation.

In the context of this specification "comprising" is to be interpreted as "including". Aspects of the invention comprising certain elements are also intended to extend to alternative embodiments "consisting" or "consisting essentially" of the relevant elements.

EXAMPLES

Chromosome extraction from periferal blood and bone marrow in order to define chromosomal break points. 12 ml of peripheral blood (PBS) or 4 ml of bone marrow (BM) are collected in eparine or in anticoaugulants other then EDTA that inhibits cells growth.

0.5, 1, 2, 3 ml of PBS or BM are cultivated in 8 ml of media (e.g. RPMI) for 24 hrs, 48 hrs and 72 hrs at 37⁰C and 5 % CO₂.

Add colchicines e.g. l μl per ml of culture. The cell suspensions are pooled together into one tube after resuspending the cell pellets in hypotonic solution. The suspension of swollen cells is centrifuged. The cell pellet is resuspended in ice-cold polyamine isolation buffer.

All chromosome suspensions are briefly centrifuged and the supernatant is filtered through a 20-μm mesh filter.

Chromosome suspention is applied to the flow cytometer.

Patients and BCR-ABL breakpoint sequencing

We monitored, for an average period of 26 months (range 12 to 42), 8 patients with CML diagnosed in the early CP. All participants gave written informed consent. Genomic junctions of BCR-ABL had been previously characterised in Mattarucchi et al. (Mattarucchi). Briefly, DNA from patients designated as 1, 2, 3, 4, 6, 8, 9 and 10 was extracted from blood or bone marrow, fragmented, ligated to adaptors, and amplified by a nested PCR using a BCR specific forward primer. Thus, genomic breakpoints were sequenced. The majority of patients were treated with IM monotherapy at a starting dose of 400 mg/day, except for patient #9 who was participating in a 800 mg/day trial. The dose for patients #6 and #10 was increased to 600 mg/day, as a consequence of suboptimal cytogenetic findings observed at 12 and 6 months, respectively.

Cytogenetic and molecular monitoring A cytogenetic analysis of bone marrow (approximately 20 metaphases for each patient) was performed before treatment using conventional QFQ-staining. Cytogenetic tests were repeated every 6 months, until a complete response was achieved. Then bone marrow metaphases were analyzed less frequently. Levels of BCR-ABL mRNA were measured upon diagnosis and approximately every 3 months thereafter. Molecular investigations were carried out at the reference laboratory of the CML network of the Italian Group of Hematological Malignancies in Adults (GIMEMA) at the Hospital of Bergamo, Italy. All analytical procedures were subject to the quality control process according to the ISO 9001 :2000 accreditation of the laboratory. The RQ-PCR protocols proposed by the EAC program were adopted, and results were reported as the ratio between the number of BCR-ABL and ABL transcripts, with this ratio expressed as a percentage (BCR-ABL/ABL) (Gabert + Beillard). Copy numbers were determined using plasmid calibrators purchased from Ipsogen (Marseille, France). The All-Prep DNA/RNA Kit (Qiagen, Hilden, Germany) was used to extract the mRNA from blood and bone marrow samples previously treated with the HetaSept gradient (Stem Cell Technologies, Vancouver, Canada) to eliminate red cells and erythroid precursors. Patient-specific genomic assays For each patient, a genomic assay was developed on the basis of his/her BCR-ABL sequence. Each assay comprised two real time reactions: one directed against the breakpoint sequence (present in 1 copy, only in leukemic cells) and a second against the BCR sequence used as control (1 copy in leukemic cells and 2 copies in normal cells). Thus, the percentage of leukemic cells (LC) was calculated using the following formula: LC = 100 ^• (2/(2ΔCt +1)), where ΔCt is the difference between the amplification cycles of the BCR-ABL and BCR reactions. Common forward primers and probes (Table 1) were used to ensure that the two reactions in each assay had similar efficiencies (i.e., > 90%). Furthermore, assays were tested to ensure high sensibility (i.e., at least 10-5 starting from 300 ng of DNA), and absence of spurious amplifications after 45 cycles. DNA was extracted with the All-Prep DNA/RNA Kit from the same samples used for molecular monitoring of mRNA, as described above. The reaction mixture contained: 12.5 μl of the TaqMan® Universal PCR MasterMix (Appliedbiosystems, Foster City, CA, USA); 900 nM of each primer; 200 nM of probe; DNA ranging from 100 ng to 300 ng depending on the sample availability; and nuclease free water up to 25 μl. The PCR thermal profile was: 2 minutes at 50 °C followed by 10 minutes at 95 °C and 45 amplification cycles (95 °C for 15 seconds and 60 °C for 60 seconds). Reactions were prepared and run in triplicate on ABI Prism 7000 SDS (Appliedbiosystems) and each experiment was repeated and confirmed a second time.

Table 1. Primer and probe sequences.

Patient ID

1 F 5 - CTGCTGCTGGGTGGTTGA-3^'

Ph-R ₅ '-GG ATTTTAGTCCTT ACTTGTTTTCTATTTCAC-3 ^'

\vt-R S'-GCCAGATCCAAGGCACAGA-3'

Probe ό-FAM-AGATGCACGGCTTC-MGB

2 F ₅ - CCCCCTTCCTGTTAGCACTTT -3^'

Ph-R 5 '- GCTGCAACAGTACAAACAGTAACCC -3 ^" wt-R 5 - CCCTAACAAGCATAGCTCTTCCTT -3'

Probe 6-FAM- ATGGGACTAGTGGACTTT -MGB

3 F 5^'- GCCCTCCTCTCCTCCAGCTA O' Ph-R 5'- AAGCCTCTGGCGTGTTTCC -3' wt-R 5 ^'- TGAGCATATGTGCAACAGTGAATG -3 ^"

Probe ₆-FAM- CACTTTTGGTCAAGCTG -MGB

4 F 5^"-TGGGACTAGTGGACTTTGGTTCA -S^"

Ph-R S^'-GTGCATGATCATCACTAGTTAAAATGTAAA -3^" wt-R 5 ' -CTAACCCACCTTGTCCACTCCT -3 ^'

Probe ό-FAM-ACAAGAGGCCCTAACAA -MGB

6 F 5'- CACAGCATACGCTATGCACATGT -S'

Ph-R 5'-GGGAAAAAATGTTTTCTCCTTATATCG -3^" wt-R 5 ' -ATAAGGTTCCAAGGACAGCAGAG -3 ^"

Probe ό-FAM-ACACACACCCCACCC -MGB

8 F 5'-TGCTCTGTGCCTTGGATCTG -3'

Ph-R 5 '-TTCGGTGTAAAATCCTTCCATACTTT -3 ^' wt-R 5'-TGCAAAACAGCTTGACCAAAA -S'

Probe 6-FAM-CCCCACTCCCGTCCT -MGB

9 F 5'- TTGTCACCTGCCTCCCTTTC O'

Ph-R 5 '- TGAACTCCTGACCTCAAGTGATCT -3 ^' wt-R 5 ^' - GAGCCCCGGAGACTCATCA -3 '

Probe ό-FAM- CGGGACAACAGAAGC -MGB

10 F 5 - CACTGGTTTGCCTGTATTGTGAA -3^' Ph-R 5 - GGACACACAGGGAACTACACTGC -3 ^' wt-R 5 - TGGGCCAAAAACATACTCATCA -3 ' Probe 6-FAM- TCCTGAGATCCCC -MGB

PRl F 5'-CCGCTGACCATCAATAAGGAAO ^' Ph-R 5 -TGCCACGCCTTCTCTTCTG-3^'

\vt-R 5'- CAAAGTCCACTAGTCCCATCAAAA -3

Probe 6-FAM-TTTCCGTGTACAGGGCA-MGB

PR2 F 5'- TTTTGGTC AAGCTGTTTTGCA-3^'

Ph-R 5'- GGC ACCAGAAGCTG AGTGAAG-3 vvt-R 5'- ACACATGTGCATAGCGTATGCTG -3

Probe ₆-FAM- TGTTGCACAT ATGCTC-MGB

For each patient-specific assay, primers are designated as follows: F, common forward primer; Ph-R, reverse primer for the selective amplification of the BCR-ABL sequence; wt-R, reverse primer for the selective amplification of BCR.

Table 2. Levels of residual disease (%) I measured by simultaneous mRNA and DNA analysis.

Patient Months of therapy from the diagnosis ID 0 3 6 12 15 18 21 24 27 30 33 42

228* 2 730* 0 050* UND* - - UND* -

1 35* 0 077* 0 004* UND* UND*

264* 1 630* 0 180* - - - - UND* - -

2 87* 1 064* 0 391* UND* -

144* - 0 920* - 0 200* 0 180* 0 060* - -

3 94* 0 801 * 0 079* 0 061 * 0 027*

64* 7 070* 1 450* 0 580* 1 660* 0 450* 0 400* 0320* -- -

4 90* 4 575* 0 672* 0418* 0 032* 0 045* 0 068* 0 024* --

92* 12 480* 7 770* 3 640* - 2 190* 7 030* 1 260* - 3 880* -

6 90* 19 669* 4 895* 3 401* 1 478* 3 567* 0 855* - 1 026* -

170* 28 220* 1 820* 0 090* 2 080* 0 040* 0 060* 0 030* UND* -- UND*

8 72* 2 228* 0 054* 0 016* 0 009* 0 006* 0 005* 0 003* 0 002* - 0 001 *

165* - 0 350* 0 230* 0 140* 1 060* 0 020* UND* - 0 260* --

9 90* 0 883* 0204* 0 128* 0 533* 0 096* 0 134* -- 0 678* -

60* - ~ 0 040* - 0 050* - - -

10 93* 0 007* 0 012*

280* - 2 900* - - 0 400* - ~ -

PRl 94* 1 805* 0212*

170* - - - 0 070* - - 0 020* -- -

PR2 65* 0 012* 0 010* --

For each patient's row, results are reported as follow: top, percentage of BCR-ABL/ABL mRNA; bottom, percentage of leukemic cells. Results at the onset are approximated to the unit. --, unavailable data; UND, undetectable levels; *, bone marrow samples; f, peripheral blood samples.

Claims

Claims

1. A method for monitoring a disease generated in a patient by a genomic translocation or providing a prognosis in relation to the disease comprising the step of: analysing an in vitro sample of at least 2,000 cells derived from said patient for: the presence of a break point in genomic DNA in the sample cells, and/or the quantity of cells with a genomic break point, wherein the method employs direct one-step analysis of DNA from said sample cells.

2. A method according to claim 1 wherein said method is suitable for analysing if: a) there are no cells containing a genomic break poiont, b) the number of cells with a genomic break point is constant in comparison to a previous analysis by the same method, c) the number of cells with a genomic break point has increased or decreased in comparison to a previous analysis by the same method..

3. A method according to claim 1 or 2 wherein said analysis is able to detect one cell containing a genomic break point in 500,000 to 1 million cells.

4. A method according to any one of claims 1 to 3 wherein 5,000 cells, 10,000 cells, 100,000 cells, 1 million 1.5 million, 2 million, 2.5 million, 3 million, 3.5 million, 4 million, 4.5 million, 5 million, 5.5 million, 6 million, 6.5 million, 7 million, 7.5 million, 8 million, 8.5 million, 9 million, 9.5 million, 10 million cells or more cells are analysed for the presence of a breakpoint.

5. A method according to any one of claims 1 to 4, wherein the method employs PCR.

6. A method according to claim 5, wherein the PCR employs patient specific primers and/or probes, for example designed based on a patient's introns

7. A method according to any one of claims 1 to 6 wherein the method is quantitative for the amount of cells containing a genomic break point in said sample.

8. A method according to any one of claims 1 to 7 wherein the disease is leukaemia, in particular chronic myeloid leukaemia or acute lymphocytic/lymphoblastic leukaemia.

9. A method according to any one of claims 1 to 8 wherein the genomic break point forms a Philadelphia chromosome.

10. A method according to any one of claims 1 to 9 which further comprises the step of quantitatively assaying the amount of mRNA from the cells containing a relevant genomic translocation.

11. A method according to any one of claims 1 to 10 comprising the further step of: discontinuing of the treatment, or modulating the dose of medicament received by a patient.

12. A kit comprising the components/reagent for an assay according to claims 1 to 11 and/or instructions on how to perform the assay.