WO2009013614A2 - Marker gene - Google Patents

Abstract

A method of aiding in prognosing chronic myeloid leukemia (CML) by determining the level of BMI-1 mRNA or protein in a sample provided from a patient. A method of increasing the life expectancy of a patient in the chronic phase of chronic myeloid leukemia comprising administering a compound which is capable of reducing the expression or function of BMI-1 mRNA or protein in the patient. A method of aiding in assessing the suitability of a patient with CML to receive an allogeneic stem cell transplant, or such a transplant after enhanced prophylaxis for graft versus host disease, comprising determining the level of BMI-1 mRNA or protein in a sample provided from a patient. Associated methods of treating a patient, depending on the outcome of such assessment are provided

Description

MARKER GENE

The present invention relates to a method of aiding in prognosing chronic myeloid leukemia (CML). The invention also provides a method of increasing the life expectancy of a patient in the chronic phase of chronic myeloid leukemia. The invention further provides a method of aiding in assessing the suitability of a patient with CML to receive an allogeneic stem cell transplant, or such a transplant after prophylaxis for graft versus host disease. The invention provides associated methods of treating a patient, depending on the outcome of such assessment.

Chronic myeloid leukemia (CML), also known as chronic myelogenous leukemia, is characterized by increased and unregulated clonal proliferation of predominantly myeloid cells in the bone marrow. Elevated levels of mature granulocytes are found in the blood.

It is generally believed that CML is caused by a specific chromosome translocation which results in part of the BCR gene from chromosome 22 being fused with the ABL gene on chromosome 9. The resulting BCR-ABL fusion gene product is an oncogene, with tyrosine kinase activity. The classic chromosomal translocation generates a 22q- or Philadelphia chromosome and a derivative 9q+ chromosome, which can be detected by routine cytogenetics, fluorescent in situ hybridisation (FISH) or by reverse transcription and polymerase chain reaction (RT-PCR) for the BCR-ABL fusion (Morgan GJ, Pratt G. Modern molecular diagnostics and the management of haematological malignancies. Clin Lab Haematol. 1998 Jun;20(3): 135-41). In some cases of CML, complex chromosomal abnormalities may mask the t(9;22) translocation, or there may be evidence of the translocation by FISH or RT-PCR in spite of normal routine karyotyping (Moloney (1987) Blood 70:905-908; Savage et al (1997) Br J Haematol 96: 1 11-116). BCR-ABL causes an increase in cell division and inhibits correct DNA repair, causing genomic instability and the acquisition of further genetic abnormalities. CML is diagnosed by detecting the (9;22) translocation. Usually, diagnosis is made in the chronic phase (CP), in which patients are often asymptomatic or have only mild symptoms. In the absence of curative treatment, the disease progresses to an accelerated phase. Criteria useful in diagnosis of the accelerated phase have been put forward by Kantarjian et al (1988) Cancer 61:1441-6; Sokal et al (1988) Semin Hematol 25: 49-61; and Vardiman, Harris and Brunning (2002) Blood 100: 2292-302. The WHO criteria (Vardiman, Harris and Brunning, supra) are widely used and include 10-19% blasts 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 patient is considered to be in the accelerated phase if any of the above is present. The final phase of CML is blast crisis, which behaves like an acute leukemia with rapid progression and short survival. Blast crisis is diagnosed if any of the following are present in a patient with CML: >20% blasts in the blood or bone marrow; large clusters of blasts in the bone marrow on biopsy; development of chloroma (solid focus of leukemia outside the bone marrow) (Cortes J, Kantarjian H. Advanced-phase chronic myeloid leukemia. Semin Hematol. 2003;40:79-86). A minority of patients are in the accelerated phase or blast crisis when they are diagnosed with CML.

Although CML eventually enters an acute phase if untreated, it is distinct from acute myeloid leukemia (AML). The latter is caused by genetic mutations which arrest differentiation of a myeloblast in the bone marrow, combined with further mutations which result in its uncontrolled growth. AML is typically fatal within weeks or months if untreated. Unlike in CML, there is no universal chromosomal abnormality underlying all AMLs. The most important prognostic factors for AML are the karyotype of the cancer cells. For example, AML with monosomy 7 has a poor prognosis, whereas AML with t(8;21) has a good prognosis (Grimwade et al (1998) Blood 92: 2322-2333). Where there are complex abnormalities, there is a poor prognosis (Grimwade et al (2001) Blood 98: 1312-1320). C/EBP-α mutations confer good prognosis in AML (Frδhling et al (2004) J Clin Oncol 22: 624-33) but were not found in the blast crisis of CML (Pabst et al (2006) Br J Haem 133: 400-2). AML can develop spontaneously, or it can evolve from pre-leukemic blood disorders such as myelodysplastic or myeloproliferative syndromes, which tend to be diseases of the elderly. CML is not known to develop from these diseases.

The duration of the chronic phase of CML varies widely between patients. The Sokal and Hasford prognostic scores (Sokal et al (1984) Blood 63: 789-799; Hasford et al (1998) J Natl Cancer Inst. 90: 850-858) proved moderately useful in predicting the duration of survival for individual patients treated with busulfan or interferon-alpha, respectively. The Sokal score also serves to discriminate survival without progression in patients treated with imatinib (Hughes et al (2003) N Engl J Med. 349: 1423-1432). Deletions adjacent to the ABL-BCR junction on the derivative 9q+ chromosome may be associated with an adverse prognosis (Huntly, Bench and Green (2003) Blood 102: 1160-1168) but only about 10% of patients with CML have this additional cytogenetic abnormality. Telomere lengths, which are already shorter in CML compared with normal cells, have been reported to be further shortened in patients in CP with early disease progression (Boultwood et al (2000) Blood 96: 358-361). Gene expression profiling of CD34+ cells collected at diagnosis of chronic phase CML patients was used to identify genes differentially expressed in "aggressive disease", in which blastic transformation (blast crisis) developed within three years, and "indolent disease" in which blastic transformation developed after seven or more years in Yong et al (2006) Blood 107: 205-12. Differential expression of genes was confirmed by quantitative RT/PCR. Low expression of CD7 in combination with high expression of proteinase 3 or elastase correlated with longer survival.

There is a need for further markers to aid the prognosis of CML in individual patients.

The present inventors found that the polycomb group gene BMI-I is a molecular marker for prognosis of CML. BMI-I is a transcriptional repressor that regulates self-renewal of normal stem cells and cancer stem cells, for example in a murine model intended to reflect AML (Lessard and Sauvageau (2003) Nature 423: 255-260). Overexpression of BMI-I can contribute to the development of various tumours, particularly lymphomas (Haupt et al (1993) Oncogene 8:3161-3164). However, if has not hitherto been implicated in the CML disease progress. Indeed, Bea et al found no evidence of BMI-I gene amplification in CML, although BMI-I gene amplifications were detected in a proportion of mantle cell lymphomas {Cancer Research (2001) 61: 2409-2412).

A preferred initial therapy for chronic phase CML is imatinib, an inhibitor of the BCR-ABL tyrosine kinase (Druker et al (2006) New Engl J Med 355: 2408-17). Other therapies include hydroxyurea or interferon-α. Before the imatinib era, chronic phase lasted on average 4 to 5 years before progressing to blastic transformation, which usually proved fatal within a few months. Thus, until recently, the median survival for patients treated predominantly with interferon- alpha was around 5 to 6 years (Kantarjian et al (2003) Cancer 97: 1033-41). Despite the very encouraging responses to imatinib, which can allow longer survival, there is good evidence that it does not eradicate all leukemic progenitor cells (Graham et al (2002) Blood 99: 319-325; Bhatia et al (2003) Blood 101: 4701-4707; Michor et al (2005) Nature 435: 1267-1270; Copland et al. Cancer Immunol Immunother. 2005;54:297-306.)

High dose radiotherapy, chemotherapy or chemoradiotherapy with marrow rescue by stem cell transplantation (SCT) is the only curative treatment for CML. The probability of survival at 5 years after allogeneic transplantation from HLA- identical donors is 60% for patients who receive transplants in chronic phase, 22% for patients in accelerated phase and 13% for patients in blast phase (see discussion in Martin et al (1988) Blood 72: 1978-1984), encouraging early transplantation for appropriate patients. However, transplantation during chronic phase is associated with 20% mortality during the first 100 days and 30% mortality during the first year, which is greater than would be expected without transplantation. The impact of imatinib on outcome, when used as an initial treatment prior to transplantation is still controversial (Deininger et al The effect of prior exposure to imatinib on transplant-related mortality. Haematologica. 2006;91 :452-9).

Thus, there is a need for prognostic markers to identify CML patients who would benefit most from allogeneic stem cell transplantation, as opposed to other forms oftreatment.

The present inventors found that BMI-I expression prior to transplantation is a predictor of outcome following allogeneic stem cell transplantation in chronic phase CML.

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

A first aspect of the invention provides a method of aiding in prognosing chronic myeloid leukemia in a patient who has chronic myeloid leukemia, the method comprising providing a sample from the patient, determining the level of BMI-I mRNA or protein in the sample and assessing whether the level is indicative of a particular outcome for the patient.

It will be appreciated that the invention of the first aspect includes a method of assessing the likely outcome of a patient who has CML, the method comprising providing a sample from the patient, determining the level of BMI-I mRNA or protein in the sample and assessing whether the level is indicative of a particular outcome for the patient. It will be appreciated that this assessment may aid prognosis, and may be used in association with other tests, or observations by the physician, in reaching a prognosis.

CML is typically diagnosed by detection of the BCR-ABL translocation by routine cytogenetics, fluorescent in situ hybridisation (FISH) or PCR (O'Brien S et al. Chronic myelogenous leukemia and myeloproliferative disease. Hematology Am Soc Hematol Educ Program. 2004:146-62).

The sample from the patient, which is analysed for expression of BMI-I mRNA or protein, may be any suitable sample. Typically, the sample contains cells of the patient, particularly hematopoietic cells. Typically, it is prepared from blood or bone marrow.

The cDNA sequence of Homo sapiens BMI-I, also known as murine leukemia viral (bmi-1) oncogene ho mo log; oncogene BMI-I; polycomb group ring finger 4; B lymphoma Mo-MLV insertion region; B lymphoma Mo-MLV insertion region 1 ho mo log; flvi-2/bmi-l, is reproduced below (NCBI Accesssion No. NM_005180).

cagcaactat gaaataatcg tagtatgaga ggcagagatc ggggcgagac aatggggatg 60 tgggcgcggg agccccgttc cggcttagca gcacctccca gccccgcaga ataaaaccga 120 tcgcgccccc tccgcgcgcg ccctcccccg agtgcggagc gggaggaggc ggcggcggcc 180 gaggaggagg aggaggaggc cccggaggag gaggcgttgg aggtcgaggc ggaggcggag 240 gaggaggagg ccgaggcgcc ggaggaggcc gaggcgccgg agcaggagga ggccggccgg 300 aggcggcatg agacgagcgt ggcggccgcg gctgctcggg gccgcgctgg ttgcccattg 360 acagcggcgt ctgcagctcg cttcaagatg gccgcttggc tcgcattcat tttctgctga 420 acgactttta actttcattg tcttttccgc ccgcttcgat cgcctcgcgc cggctgctct 480 ttccgggatt ttttatcaag cagaaatgca tcgaacaacg agaatcaaga tcactgagct 540 aaatccccac ctgatgtgtg tgctttgtgg agggtacttc attgatgcca caaccataat 600 agaatgtcta cattccttct gtaaaacgtg tattgttcgt tacctggaga ccagcaagta 660 ttgtcctatt tgtgatgtcc aagttcacaa gaccagacca ctactgaata taaggtcaga 720 taaaactctc caagatattg tatacaaatt agttccaggg cttttcaaaa atgaaatgaa 780 gagaagaagg gatttttatg cagctcatcc ttctgctgat gctgccaatg gctctaatga 840 agatagagga gaggttgcag atgaagataa gagaattata actgatgatg agataataag 900 cttatccatt gaattctttg accagaacag attggatcgg aaagtaaaca aagacaaaga 960 gaaatctaag gaggaggtga atgataaaag atacttacga tgcccagcag caatgactgt 1020 gatgcactta agaaagtttc tcagaagtaa aatggacata cctaatactt tccagattga 1080 tgtcatgtat gaggaggaac ctttaaagga ttattataca ctaatggata ttgcctacat 1140 ttatacctgg agaaggaatg gtccacttcc attgaaatac agagttcgac ctacttgtaa 1200 aagaatgaag atcagtcacc agagagatgg actgacaaat gctggagaac tggaaagtga 1260 ctctgggagt gacaaggcca acagcccagc aggaggtatt ccctccacct cttcttgttt 1320 gcctagcccc agtactccag tgcagtctcc tcatccacag tttcctcaca tttccagtac 1380 tatgaatgga accagcaaca gccccagcgg taaccaccaa tcttcttttg ccaatagacc 1440 tcgaaaatca tcagtaaatg ggtcatcagc aacttcttct ggttgatacc tgagactgtt 1500 aaggaaaaaa attttaaacc cctgatttat atagatatct tcatgccatt acagctttct 1560 agatgctaat acatgtgact atcgtccaat ttgctttctt ttgtagtgac attaaatttg 1620 gctataaaag atggactaca tgtgatactc ctatggacgt taattgaaaa gaaagattgt 1680 tgttataaag aattggtttc ttggaaagca ggcaagactt tttctctgtg ttaggaaaga 1740 tgggaaatgg tttctgtaac cattgtttgg atttggaagt actctgcagt ggacataagc 1800 attgggccat agtttgttaa tctcaactaa cgcctacatt acattctcct tgatcgttct 1860 tgttattacg ctgttttgtg aacctgtaga aaacaagtgc tttttatctt gaaattcaac 1920 caacggaaag aatatgcata gaataatgca ttctatgtag ccatgtcact gtgaataacg 1980 atttcttgca tatttagcca ttttgattcc tgtttgattt atacttctct gttgctacgc 2040 aaaaccgatc aaagaaaagt gaacttcagt tttacaatct gtatgcctaa aagcgggtac 2100 taccgtttat tttactgact tgtttaaatg attcgctttt gtaagaatca gatggcatta 2160 tgcttgttgt acaatgccat attggtatat gacataacag gaaacagtat tgtatgatat 2220 atttataaat gctataaaga aatattgtgt ttcatgcatt cagaaatgat tgttaaaatt 2280 ctcccaactg gttcgacctt tgcagatacc cataacctat gttgagcctt gcttaccagc 2340 aaagaatatt tttaatgtgg atatctaatt ctaaagtctg ttccattaga agcaattggc 2400 acatctttct atactttata tacttttctc cagtaataca tgtttacttt aaaaattgtt 2460 gcagtgaaga aaaaccttta actgagaaat atggaaaccg tcttaatttt ccattggcta 2520 tgatggaatt aatattgtat tttaaaaatg catattgatc actataattc taaaacaatt 2580 ttttaaataa accagcaggt tgctaaaaga aggcatttta tctaaagtta ttttaatagg 2640 tggtatagca gtaattttaa atttaagagt tgcttttaca gttaacaatg gaatatgcct 2700 tctctgctat gtctgaaaat agaagctatt tattatgagc ttctacaggt atttttaaat 2760 agagcaagca tgttgaattt aaaatatgaa taaccccacc caacaatttt cagtttattt 2820 tttgctttgg tcgaacttgg tgtgtgttca tcacccatca gttatttgtg agggtgttta 2880 ttctatatga atattgtttc atgtttgtat gggaaaattg tagctaaaca tttcattgtc 2940 cccagtctgc aaaagaagca caattctatt gctttgtctt gcttatagtc attaaatcat 3000 tacttttaca tatattgctg ttacttctgc tttctttaaa aatatagtaa aggatgtttt 3060 atgaagtcac aagatacata tatttttatt ttgacctaaa tttgtacagt cccattgtaa 3120 gtgttgtttc taattataga tgtaaaatga aatttcattt gtaattggaa aaaatccaat 3180 aaaaaggata ttcatttaga aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3240 aaaaaaaaaa a 3251

It will be appreciated that the sequence of bases in the mRNA sequence corresponding to the above cDNA sequence would be the same except that uracil would replace thymidine. A BMI-I cDNA sequence published by different researchers has NCBI Accession No. BCOl 1652. Further mRNA and cDNA sequences of BMI-I may be known to the person of skill in the art, reflecting allelic variation within the human population, or mutation. In analysing the level of expression of BMI-I mRNA, all such mRNA sequences are encompassed.

The level of mRNA may be measured by any method known in the art. Quantitative methods are preferred, such as reverse transcription and quantitative real time polymerase chain reaction. Suitable semi-quantitative methods are reverse transcription PCR or northern blotting. Oligonucleotide micro-array technology may also be used to quantify BMI-I expression, for example in the context of an expression array for CML. An example of the use of an oligonucleotide microarray in CML, and genes that it would be useful to include in a custom microarray for prognosing CML, is described in Yong AS, et al. Molecular profiling of CD34+ cells identifies low expression of CD7, along with high expression of proteinase 3 or elastase, as predictors of longer survival in patients with CML. Blood. 2006; 107:205-212. Preferably, levels of mRNA are determined by reverse transcription and quantitative real time polymerase chain reaction (Q-RT7PCR). Real-time detection of PCR products relies on the inclusion in the reaction of a fluorescent molecule that reports an increase in the amount of DNA with a proportional increase in fluorescent signal. Suitable fluorescent chemistries employed for this purpose may include DNA-binding dyes and fluorescently labeled sequence specific primers or probes. Specialized thermal cyclers equipped with fluorescence detection modules are used to monitor the fluorescence as amplification occurs. During the early cycles of PCR amplification of the template, the quantity of product increases exponentially. Initially, increases in fluorescence are below the level of detection. However, eventually, enough DNA product accumulates to yield a detectable fluorescent signal. The cycle number at which this occurs is called the threshold cycle, or C_T. Since the C_T value is measured in the exponential phase when reagents are not limited, Q- RTVPCR can be used to reliably and accurately calculate the initial amount of template present in the reaction. Guidelines for the use and optimisation of Q- RTVPCR are described in detail on the Bio-Rad website at http://www.bio- rad.com/pdf/Bulletin_5279B.pdf.

Suitable fluorescent chemistries are: 1) DNA-binding dyes such as SYBR Green I), and 2) dye-labeled, sequence-specific oligonucleotide primers or probes such as molecular beacons, TaqMan^®, hybridization, and Eclipse probes, and Amplifluor, Scorpions, LUX, and BD QZyme primers. The use of these chemistries and guidance for the design of suitable primers and probes is described in http://www.bio-rad.com/pdf/Bulletin_5279B.pdf.

Preferably, Q-RT/PCR is performed according to the TaqMan^® method, which utilises a probe that is fluorescently labelled with a reporter dye and a quenching dye.

The Taqman^® method is now well known in the art and has been described, for example, by Van der Velden et al (2003) Leukemia 17: 1013-3, Gabert et al (2005) Leukemia 17: 2318-57 and Branford et al (2006) Methods MoI Med 125: 69-92.

Briefly, this method is based on the 5'-3' exonuc lease activity of Taq DNA polymerase, which results in cleavage of fluorescent dye-labelled probes during PCR; the intensity of fluorescence is then measured by a detection system. The probe binds to the cDNA template at a location between the binding sites of the two PCR primers and usually has a melting temperature around 10°C higher than that of the primers. The probe has two fluorescent tags attached to it. One is a reporter dye, such as 6-carboxyfluorescein (FAM), which has its emission spectra quenched due to the spatial proximity of a second fluorescent dye, 6-carboxy- tetramethyl-rhodamine (TAMRA). Degradation of the probe, by the Taq DNA polymerase, frees the reporter dye from the quenching activity of TAMRA and thus the fluorescent activity increases with an increase in cleavage of the probe, which is proportional to the amount of PCR product formed. The ABI Prism 7700 is a laser-coupled spectrophotometer which is suitable for monitoring the fluorescence output of TaqMan^® performed in a microtitre plate format in realtime.

The intersection between the amplification plot and the threshold, where the threshold is defined as 10 times the standard deviation of the background fluorescence intensity and which is typically measured between cycle 3 and 15, is known as the cycle threshold, or Ct, value. The Ct value is directly related to the amount of PCR product and therefore related to the initial amount of target DNA present in the PCR reaction.

Typically, in methods of quantifying mRNA, total RNA is extracted from the cells in the sample and preferably treated with DNAse to eliminate genomic DNA (Maurisse et al., Gel purification of genomic DNA removes contaminating small DNA fragments interfering with polymerase chain reaction analysis of small fragment homologous replacement. Oligonucleotides. 2006;16:375-86). Typically, RNA is reverse-transcribed into cDNA and the level of mRNA indirectly measured by quantitative real time polymerase chain reaction (Q- RT/PCR) using the cDNA as template (Provenzano M, Mocellin S. Complementary techniques: validation of gene expression data by quantitative real time PCR. Adv Exp Med Biol. 2007;593:66-73). It is typical to express the level of the mRNA species of interest, in this case BMI-I, with reference to the level of mRNA of a gene, the level of expression of which is thought to be relatively constant between cells. This allows for variations between samples in the quantity of cells, and the efficiency of RNA extraction and reverse- transcription, to be normalised. Where the TaqMan^® method is used, it is possible to perform multiplex Q-RT/PCR, in which the levels of more than one cDNA species, and typically up to five cDNA species, can be quantified in the same reaction. The choice of such a reference gene may depend on the type of cells in the sample. Typically, a house-keeping gene is selected, preferably GAPDH, beta-2-microglobulin or beta glucuronidase (GUS). ABL or BCR may also be used (Gabert et al. 1 Standardization and quality control studies of 'real- time¹ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program. Leukemia. 2003;17:2318-57).

Typically, random hexameric oligonucleotide primers, with or without oligo-dT oligonucleotide primers, are used in reverse-transcribing the RNA to prepare cDNA. This allows for reverse-transcription of the RNA to be relatively independent of nucleotide sequence. Thus, the cDNA sample so obtained should be suitable for performing PCR or real time PCR to identify and/or quantify a variety of cDNA species. Alternatively, the skilled person may design oligonucleotide primers specifically for reverse-transcribing the BMI-I mRNA into cDNA, together with primers for specifically reverse-transcribing other mRNA species of interest, such as GAPDH.

One or more, and typically two oligonucleotides are required for the quantitative PCR step in Q-RT/PCR. A skilled person can readily design suitable oligonucleotides for the amplification of BMI-I cDNA, or for the amplification of other cDNAs such as those of GAPDH, beta-2-microglobulin, GUS, ABL or BCR, and, where appropriate, suitable probes for detection of the PCR product in real time.

Where DNA-binding dyes such as SYBR Green are used to quantify the PCR product in real-time, an oligonucleotide probe may not be required. A pair of oligonucleotide primers is used to amplify a region of the cDNA of interest, such as BMI-I cDNA. Then the probe binds non- specifically to all double-stranded DNA products.

Suitably, where the TaqMan^® method is used, a pair of oligonucleotide primers are required to amplify a region of the cDNA of interest and a probe labelled with a reporter dye and a quenching dye as described above. Suitable primer and probe combinations are available from Applied Biosystems as "assays-on- demand" or "gene-expression-on-demand". A suitable combination for measuring BMI-I cDNA by Q-RT/PCR is supplied by Applied Biosystems as cat. no. HsOOl 8041 l_ml. The probe binds at the exon 3-4 boundary and the amplicon length 105bp. A suitable combination for measuring GAPDH cDNA by Q-RT/PCR is also supplied by Applied Biosystems.

As an alternative to measuring the level of BMI-I mRNA in the sample, the level of BMI-I protein may be measured by any suitable means. One convenient way of measuring the level of BMI-I protein in the sample is to make use of a reagent which can identify BMI-I protein. Conveniently, the reagent is one which binds to BMI-I protein, but it may be any other type of suitable reagent.

Reagents which bind to BMI-I protein include antibodies and peptides selected from a combinatorial or phage display library. By the term "antibodies" we include whole antibodies which bind to BMI-I protein but also fragments of antibodies which bind BMI-I protein such as Fv, Fab and F(ab)₂ fragments as well synthetic antibodies or antibody fragments such as single chain Fv (scFv) molecules and domain antibodies (dAbs). The antibody fragments and synthetic antibodies retain antigen binding activity (and usually contain some or all of the complementarity determining regions (CDRs) of a parent antibody molecule). Antibodies for BMI-I protein may be made using well known technology such as the hybridoma method for making monoclonal antibodies, and phage display techniques for making synthetic antibody fragments. Suitable methods for the production and use of antibodies are described and referred to in "Using antibodies: A laboratory manual", Ed Harlow and David Lane, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1999. A suitable anti-BMI-1 monoclonal antibody is available from Upstate Signaling Solutions, Lake Placid, NY. Its use in detecting BMI-I in human cells is described in Mihara et al (2006) Blood 107:305-308).

Quantitative methods of determining the level of BMI-I protein are preferred. Conveniently, the level of BMI-I protein in the sample is measured using an immunoassay. The antibody selective for BMI-I protein may itself be labelled, for example with a radioactive label or a fluorescence label or with an enzyme. Alternatively, it is detected with a secondary antibody, which is labelled and which binds the antibody selective for BMI-I protein. BMI-I is an intracellular protein. Accordingly, it is preferred to measure the level of BMI-I protein on cells in the sample by flow cytometry, for example using a fluorescence activated cell sorting (FACS). A suitable method is described in Mihara et al (2006) supra. An alternative immunoassay is an ELISA. BMI-I should be extracted from lysed cells for detection by ELISA. Immunoassays are well known in the art (see, for example, Immunoassays: A practical approach. James P. Gosling (ed), Oxford University Press, 2000, ISB4 0-19-963710-5). Semi-quantitative methods may also be used, such as western blotting.

In assessing whether the level of BMI-I mRNA or protein is indicative of a particular outcome for the patient, an elevated level of BMI-I mRNA or protein is indicative of a poor outcome.

The level of BMI-I mRNA or protein that is indicative of a poor prognosis may vary depending on the type of patient. The level may be determined by comparing levels in CML patients who fare well and those who have a poor outcome. The inventors have found that a level of BMI-I mRNA of greater than the median for a cohort of patients at diagnosis of CML in the chronic phase is indicative of a poor outcome, as described in the Examples. A level of BMI-I protein of greater than the median for a cohort of patients at diagnosis of CML in the chronic phase is also indicative of a poor outcome. It may be appropriate to set a different threshold level of BMI-I mRNA or protein to aid in categorising patient groups according to prognosis. For example, a level of BMI-I mRNA or protein that is greater than one, or greater than two standard deviations (SD), above the mean or median level of BMI-I mRNA in a population of CML patients at diagnosis who fare well may be indicative of a poor outcome. A level of BMI-I mRNA or protein that is above the third quartile (upper quartile) in a population of CML patients at diagnosis may be indicative of a poor outcome. A level of BMI-I mRNA or protein that is below the first quartile (lower quartile) in a population of CML patients at diagnosis may be indicative of a good outcome. The availability of patient samples collected at diagnosis of CML, and preserved, for example by cryopreservation, allows the level of BMI-I to be correlated with disease outcome retrospectively. Such analyses, conducted on a large group of patients, will allow the correlation between BMI mRNA or protein level and prognosis to be refined.

It will be appreciated that not all patients are diagnosed with CML in chronic phase. The method is most suitable for aiding in the prognosis of those patients who are diagnosed in chronic phase.

In the context of prognosis of CML, a "poor prognosis" may include a prognosis of "aggressive disease". Patients with aggressive disease typically develop blast crisis early after diagnosis, such as in fewer than three years from diagnosis. A "good prognosis" may include a prognosis of "indolent disease". Typically, patients with indolent disease develop blast crisis more than seven years after diagnosis. An "intermediate prognosis" may include a prognosis of "intermediate disease", in which patients typically develop blast crisis between three and seven years after diagnosis in chronic phase. The above indications of time to blast crisis in indolent, intermediate and aggressive disease are based on patients who do not receive stem cell transplants, but who instead receive drug therapy. It will be appreciated that the development of new drugs, such as imatinib and other tyrosine kinase inhibitors, may improve the life expectancy of patients treated with such drugs. Thus, the definitions of indolent, intermediate and aggressive disease, particularly with reference to the time between diagnosis in chronic phase and blast crisis, may vary. The average duration that a person has had CML before diagnosis in chronic phase may also vary, having a consequent effect on the average duration of the interval between diagnosis in chronic phase and blast crisis.

It will be appreciated that treatment decisions may differ depending on the level of BMI-I or protein, and other factors which contribute to the prognosis. For example, follow-up consultations may be given more frequently to those patients with a high level of BMI-I mRNA or protein, compared to the typical frequency of consultation for CML patients. For example, patients with high BMI-I may receive follow-up consultations approximately every three months, instead of approximately every six months. Such patients would also be candidates to receive a combination of therapies rather than a single therapy. For example, they may be treated with imatinib and a further therapy, such as interferon-alpha or hydroxyurea.

In the method of the first aspect of the invention, the sample from the patient is preferably a CD34⁺ cell sample or a peripheral blood mononuclear cell (PBMC) sample. A CD34⁺ cell may be obtained from peripheral blood, conveniently following leukapheresis. For example, CD34⁺ cells may be obtained from peripheral blood by binding to immmuno magnetic beads (MiniMACS; Miltenyi- Biotech, Bergisch-Gerbach, Germany.) It is particularly preferred that the sample is a PBMC sample as PBMCs are readily and cheaply obtainable from blood. At presentation (diagnosis) of chronic phase CML the PBMCs are essentially composed of a majority, typically over 80% leukaemic cells, and any measurable gene expression reflects expression in the CML cells. Similarly, CD34⁺ cells obtained from blood at diagnosis of chronic phase CML are typically at least 80-95% leukaemic cells. Other suitable cell samples are bone marrow cells, or susbsets of mononuclear cells obtained from bone marrow or blood cells. Preferably, the sample is collected from the patient at the time of diagnosis of CML. It is typical to collect blood for assessing blood cell counts, which often gives a preliminary indication that the patient might have CML. Blood and/or bone marrow cells are generally assessed for the presence of the BCR-ABL translocation, in order to arrive at a definite diagnosis. Typically, blood and/or bone marrow cells are collected when the patient is first referred to a specialist doctor, such as a haematologist or oncologist (Kantarjian et al, Diagnosis and management of chronic myeloid leukemia: a survey of American and European practice patterns. Cancer. 2007;l 09: 1365-75). However, even if the sample is collected subsequently, the BMI-I mRNA or protein level is still useful in aiding in prognosis.

The method of the first aspect of the invention may further comprise determining the levels of one or more further CML markers in a sample from the patient and assessing whether the levels of said further marker or markers is indicative of a particular outcome for the patient. For example, genes identified as being differentially expressed in "aggressive disease" versus "indolent disease" by Yong et al (2006) Blood 107: 205-12, are suitable for use as additional CML markers. In particular, low expression of CD7, and high expression of proteinase 3 or elastase are correlated with longer survival. Other suitable CML markers are described in Radich et al (2006) froc Natl Acad Sci USA 103: 2794-2799. Preferably, the level of BMI-I mRNA or protein and the level of the one or more CML markers are all taken into account when assessing whether the levels are indicative of a particular outcome for the patient. Preferably, the further marker is proteinase-3 (PR3). The inventors found that the combination of low BMI-I and high PR3 expression is a strong independent marker associated with significantly longer overall survival, as described in the Examples. Methods suitable for determining the level of the further marker in the sample from the patient are as described above in relation to determining the level of BMI-I mRNA or protein, except that the reagent must be suitable for detecting the further marker instead of BMI-I. Suitable reagents, such as oligonucleotides and antibodies, may be developed using the general methods described above. A suitable primers and probe combination for quantifying PR-3 cDNA by Q- RT/PCR according to the TaqMan^® method is available from Applied Biosystems as cat. no. HsOOl 6052 I mI. The probe binds at the exon 1-2 boundary and the amplicon length is 1 lObp.

A second aspect of the invention provides a use of a reagent which selectively identifies BMI-I mRNA or protein in the prognosis of CML in a patient.

In the context of the reagent, "selectively identifies" indicates that the reagent identifies BMI-I mRNA or protein, directly or indirectly, substantially without falsely identifying other molecules as BMI-I mRNA or protein in the sample in which the target is presented. Typically, the sample is a sample provided from the patient as described in relation to the first aspect, or a preparation prepared from it. The sample or preparation may contain proteins in addition to BMI-I protein and/or mRNA species in addition to BMI-I mRNA. A suitable preparation is one prepared for Q-RT/PCR, for example as described above. The reagent typically recognises a target molecule. In the context of identifying BMI- 1 mRNA or protein, the target may be the BMI-I mRNA or protein, or it may be a related molecule such as a cDNA reverse transcribed from the BMI-I mRNA. It is preferred that the reagent identifies the BMI-I cDNA. In relation to nucleic acid based reagents, selectivity corresponds to an ability to selectively hybridize to a target species, in this case BMI-I mRNA or, preferably, cDNA. By selectively hybridize is meant that the nucleic acid has sufficient nucleotide sequence similarity with the target species that it can hybridise under moderately or highly stringent conditions. As is well known in the art, the stringency of nucleic acid hybridisation depends on factors such as length of nucleic acid over which hybridisation occurs, degree of identity of the hybridizing sequences and on factors such as temperature, ionic strength and GC or AT content of the sequence. Thus, any nucleic acid that is capable of selectively hybridising as said is useful in the practice of the invention. Nucleic acids which can selectively hybridise to the said human nucleic acid include nucleic acids which have > 95 % sequence identity, preferably those with > 98 %, more preferably those with > 99 % sequence identity, over at least a portion of the nucleic acid with the said human nucleic acid.

Suitable reagents are disclosed above, and it is particularly preferred to use one or more oligonucleotides to identify BMI-I mRNA in the prognosis of CML in a patient. Suitably, the target recognised by the one or more oligonucleotides is

BMI-I cDNA. Hence the BMI-I mRNA is identified indirectly. Preferably, the one or more oligonucleotides is suitable for use in Q-RT/PCR. Thus, the invention includes a reagent which selectively identifies BMI-I mRNA or protein, such as one or more oligonucleotides, for use in prognosing CML.

Typically, the reagent is used in the methods described above.

In a preferred embodiment, this aspect of the invention also includes the use of a reagent which selectively identifies BMI-I mRNA or protein and use of a reagent that selectively identifies a further chronic myeloid leukemia marker in the prognosis of chronic myeloid leukemia in a patient. Suitable reagents that selectively identify the further CML marker are described above, but conveniently the reagent is one or more oligonucleotides. It is preferred if the further CML marker is PR3. Thus, conveniently, the invention includes one or more oligonucleotides suitable for quantifying BMI-I mRNA and one or more oligonucletoides suitable for quantifying PR3 both for use in prognosing CML in a patient. It is particularly preferred if the one or more oligonucleotides suitable for quantifying BMI-I mRNA and the one or more oligonucletoides suitable for quantifying the further CML marker are used in Q-RT/PCR for prognosing CML.

The invention also includes, as a third aspect, the use of a reagent which selectively identifies BMI-I mRNA or protein in the manufacture of a composition for prognosing CML in a patient. The composition comprises a reagent which is used to prognose CML in a patient. Typically, the reagent which selectively identifies BMI-I mRNA or protein is a reagent as described above. A fourth aspect of the invention provides a method of increasing the life expectancy of a patient in the chronic phase of CML comprising administering an agent which is capable of reducing the expression or function of BMI-I mRNA or protein in the patient.

By an 'agent' we include all chemical entities, for example oligonucleotides, polynucleotides, polypeptides, peptidomimetics and drug molecules which are small compounds. Thus, the invention provides an agent capable of inhibiting the biological activity of BMI-I directly (for example, by reducing the biological activity of the protein) or indirectly (for example, by reducing expression of the BMI-I mRNA or protein). Preferably, expression of mRNA is reduced in the patient, for example by antisense RNA or siRNA.

Liu, Andrews and Tollefsbol (2006) Oncogene 25: 4370-4375 describe a suitable short interfering (siRNA) molecule for knock-down of BMI-I expression. They showed that treatment of various human cancer cells by RNAi to knock-down BMI-I increased apoptosis and delayed cell cycle progression, but had minimal effect on survival of normal cells. Synthesis of other suitable RNAi molecules for use with the present invention can be effected as follows. First, the sequence of the mRNA molecule encoding BMI-I is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex (Tuschl, ChemBiochem. 2:239-245). It will be appreciated, however, that siRNAs directed at untranslated regions may also be effective.

Second, potential target sites are compared to an appropriate human genomic database using sequence alignment software, such as the BLAST (www.ncbi.nlm.nih.gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out. Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55%. Several target sites are preferably selected along the length of the BMI-I mRNA for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene. Advantageously, the siRNA molecule is 19 to 23 nucleotides in length.

In an alternative preferred embodiment, the agent is an antisense oligonucleotide.

The design of antisense molecules which can be used to decrease efficiently the level or activity of BMI-I requires consideration of two aspects important to the antisense approach. The first aspect is delivery of the oligonucleotide into the cytoplasm of the CML cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof.

The prior art teaches a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types (for example, see Luft, 1998, J MoI Med 76:75-6; Kronenwett et al, 1998, Blood 91:852-62; Rajur et al, 1997, Bioconjug Chem 8:935-40; Lavigne et al, 1997, Biochem Biophys Res Commun 237:566-71; Aoki et al., 1997, Biochem Biophys Res Commun 231:540-5).

In addition, algorithms for identifying those sequences with the highest predicted binding affinity for the target mRNA based on a thermodynamic cycle that accounts for the energetic of structural alternations in both the target mRNA and the oligonucleotide are available (for example, see Walton et al, 1999, Biotechnol Bioeng 65: 1 -9).

Several approaches for designing and predicting efficiency of specific oligonucleotides using an in vitro system are also known (for example, see Matveeva et al, 1998, Nature biotechnology 16:1374-1375).

Several clinical trials have demonstrated safety, feasibility and activity of antisense oligonucleotides. Antisense oligonucleotides suitable for the treatment of cancer have been successfully used (Holmlund et al., 1999, Curr Opin MoI Ther 1:372-85; Gerwitz, 1999, Curr Opin MoI Ther 1:297-306). More recently, antisense-mediated suppression of human heparanase gene expression has been reported to inhibit pleural dissemination of human cancer cells in a mouse model (Uno et al, 2001, Cancer Res 61:7855-60).

Advantageously, the antisense oligonucleotide is 15 to 35 bases in length. For example, 20-mer oligonucleotides have been shown to inhibit the expression of the epidermal growth factor receptor mRNA (Witters et al, Breast Cancer Res Treat 53:41-50 (1999)) and 25-mer oligonucleotides have been shown to decrease the expression of adrenocorticotropic hormone by greater than 90% (Frankel et al, J Neurosurg 91 :261-7 (1999)). However, it is appreciated that it may be desirable to use oligonucleotides with lengths outside this range, for example 10, 1 1, 12, 13, or 14 bases, or 36, 37, 38, 39 or 40 bases.

It will be further appreciated by person skilled in the art that oligonucleotides are subject to being degraded or inactivated by cellular endogenous nucleases. To counter this problem, it is possible to use modified oligonucleotides, e.g. having altered internucleotide linkages, in which the naturally occurring phosphodiester linkages have been replaced with another linkage. For example, Agrawal et al (1988) Proc. Natl. Acad. Sci. USA 85, 7079-7083 showed increased inhibition in tissue culture of HIV-I using oligonucleotide phosphoramidates and phosphorothioates. Sarin et al (1988) Proc. Natl. Acad. Sci. USA 85, 7448-7451 demonstrated increased inhibition of HIV-I using oligonucleotide methylphosphonates. Agrawal et al (1989) Proc. Natl. Acad. Sci. USA 86, 7790- 7794 showed inhibition of HTV-I replication in both early-infected and chronically infected cell cultures, using nucleotide sequence-specific oligonucleotide phosphorothioates. Leither et al (1990) Proc. Natl. Acad. Sci. USA 87, 3430-3434 report inhibition in tissue culture of influenza virus replication by oligonucleotide phosphorothioates.

Oligonucleotides having artificial linkages have been shown to be resistant to degradation in vivo. For example, Shaw et al (1991) in Nucleic Acids Res. 19, 747- 750, report that otherwise unmodified oligonucleotides become more resistant to nucleases in vivo when they are blocked at the 3 ' end by certain capping structures and that uncapped oligonucleotide phosphorothioates are not degraded in vivo.

A detailed description of the H-phosphonate approach to synthesising oligonucleoside phosphorothioates is provided in Agrawal and Tang (1990) Tetrahedron Letters 31, 7541-7544, the teachings of which are hereby incorporated herein by reference. Syntheses of oligonucleoside methylphosphonates, phosphorodithioates, phosphoramidates, phosphate esters, bridged phosphoramidates and bridge phosphorothioates are known in the art. See, for example, Agrawal and Goodchild (1987) Tetrahedron Letters 28, 3539; Nielsen et al (1988) Tetrahedron Letters 29, 2911; Jager et al (1988) Biochemistry 27, 7237; Uznanski et al (1987) Tetrahedron Letters 28, 3401; Bannwarth (1988) HeIv. Chim. Acta. 71, 1517; Crosstick and VyIe (1989) Tetrahedron Letters 30, 4693; Agrawal et al (1990) Proc. Natl. Acad. Sci. USA 87, 1401-1405, the teachings of which are incorporated herein by reference. Other methods for synthesis or production also are possible. In a preferred embodiment the oligonucleotide is a deoxyribonucleic acid (DNA), although ribonucleic acid (RNA) sequences may also be synthesised and applied.

The oligonucleotides useful in the invention preferably are designed to resist degradation by endogenous nucleolytic enzymes. In vivo degradation of oligonucleotides produces oligonucleotide breakdown products of reduced length. Such breakdown products are more likely to engage in non-specific hybridisation and are less likely to be effective, relative to their full-length counterparts. Thus, it is desirable to use oligonucleotides that are resistant to degradation in the body and which are able to reach the targeted cells. The present oligonucleotides can be rendered more resistant to degradation in vivo by substituting one or more internal artificial internucleotide linkages for the native phosphodiester linkages, for example, by replacing phosphate with sulphur in the linkage. Examples of linkages that may be used include phosphorothioates, methylphosphonates, sulphone, sulphate, ketyl, phosphorodithioates, various phosphoramidates, phosphate esters, bridged phosphorothioates and bridged phosphoramidates. Such examples are illustrative, rather than limiting, since other internucleotide linkages are well known in the art. The synthesis of oligonucleotides having one or more of these linkages substituted for the phosphodiester internucleotide linkages is well known in the art, including synthetic pathways for producing oligonucleotides having mixed internucleotide linkages.

Oligonucleotides can be made resistant to extension by endogenous enzymes by "capping" or incorporating similar groups on the 5 ' or 3 ' terminal nucleotides. A reagent for capping is commercially available as Amino-Link II™ from Applied Bio Systems Inc, Foster City, CA. Methods for capping are described, for example, by Shaw et al (1991) Nucleic Acids Res. 19, 747-750 and Agrawal et al (1991) Proc. Natl. Acad Sci. USA 88(17), 7595-7599.

A further method of making oligonucleotides resistant to nuclease attack is for them to be "self-stabilised" as described by Tang et al (1993) Nucl. Acids Res. 21, 2729- 2735. Self-stabilised oligonucleotides have hairpin loop structures at their 3' ends, and show increased resistance to degradation by snake venom phosphodiesterase, DNA polymerase I and foetal bovine serum. The self-stabilised region of the oligonucleotide does not interfere in hybridisation with complementary nucleic acids, and pharmacokinetic and stability studies in mice have shown increased in vzvo persistence of self-stabilised oligonucleotides with respect to their linear counterparts. Methods for targeting agents to particular cell types, such as cancer cells, are well known in the art (for example see Vasir & Labhasetwar, 2005, Technol Cancer Res Treat. 4(4):363-74; Brannon-Peppas & Blanchette, 2004, Adv Drug Deliv Rev. 56(l l): 1649-59 and Zhao & Lee, 2004, Adv Drug Deliv Rev. 56(8):1193- 204).

A fifth aspect of the invention provides a method of aiding in assessing a patient having chronic myeloid leukemia for suitability to receive an allogeneic stem cell transplant, the method comprising providing a sample from the patient prior to starting the conditioning or preparative regimen for transplantation, determining the level of BMI-I mRNA or protein in the sample and assessing whether the level is indicative of suitability to receive an allogeneic stem cell transplant.

A sixth aspect of the invention provides a method of treating a patient with chronic myeloid leukemia comprising:

(i) providing a sample from the patient;

(ii) determining the level of BMI-I mRNA or protein in the sample;

(iii) assessing whether the level of BMI-I mRNA or protein in the sample is indicative of suitability to receive an allogeneic stem cell transplant; and,

(iv) if the level of BMI-I mRNA or protein in the sample is indicative that the patient may be suitable to receive an allogeneic stem cell transplant, performing an allogeneic stem cell transplant; and if the level of BMI-I mRNA or protein in the sample is not indicative that the patient may be suitable to receive an allogeneic stem cell transplant, treating the patient with an alternative therapy.

In the fifth and sixth aspects, the patient sample is provided and the level of BMI- 1 mRNA or protein quantified as described above in relation to the first and second aspects. By a patient's "suitability to receive an allogeneic stem cell transplant" we include the patient's likelihood of benefiting from an allogeneic stem cell transplant (SCT) as opposed to some other type of therapy for CML, in the context of economic and other considerations. It will be appreciated that various _j factors must be considered in deciding on an appropriate course of therapy for a particular patient. Goker et al (2001) Exp Hematol 29: 259-277 reviews risk factors for development of graft versus host disease, which is a significant cause of mortality and morbidity in transplant recipients. Risk factors include age, female donor with previous pregnancies, patient and donor cytomegalovirus serostatus, patient and donor ABO incompatibility, disease status at time of transplant (remission vs. relapsed or advanced), origin stem cell source, HLA compatibility, GVHD prophylaxis regimens, etc. The age profile of the patient is significant. Allogeneic SCT is not usually recommended for elderly patients due to excessive mortality. The typical median age profile of adult patients diagnosed with CML is 57 (range 18-81) with 60% aged less than 60 (Kantarjian et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med. 2002 28;346:645-52.) Allogeneic SCT may be appropriate for patients up to the age of 55 to 60 years, although where non-myeloablative or reduced intensity conditioning regimes are used, allogeneic SCT may be appropriate for individuals over the age of 55 to 60 years (Wallen et al. Ablative allogeneic hematopoietic cell transplantation in adults 60 years of age and older. J Clin Oncol. 2005;23:3439-46; Crawley et al. Outcomes of reduced-intensity transplantation for chronic myeloid leukemia: an analysis of prognostic factors from the Chronic Leukemia Working Party of the EBMT. Blood. 2005;106:2969-76). A suitable related or unrelated donor must also be available to provide stem cells that arefully or partially HLA-identical to those of the recipient. Preferably the donor is a sibling of the recipient, although the donor might another member of the recipient's family or be unrelated to the recipient. These and other factors may be fed into a scoring system known as EBMT-Gratwohl to arrive at a patient-specific risk assessment (Gratwohl et al (1988) Lancet 352: 1087-1092; Passweg et a\ (2004) Br J Haematol 125: 613- 620). Thus, the EMBT-Gratwohl score may be combined with the assessment of patient suitability based on the level of BMI-I mRNA or protein to make an overall assessment. Other factors may also be considered in the assessment and decision- making process. For example, the psychological benefit of the prospect of a cure from receiving allogeneic SCT as compared to the alternative of a lifetime of drug therapy which may not offer the prospect of cure, may be considered for individual patients. Societal factors may also be considered, such as economic factors. For example, the cost of allogeneic SCT may be less than life long imatinib therapy and this might be reflected in a preference for allogeneic SCT (Gratwohl et al.; Hematopoietic stem cell transplants for chronic myeloid leukemia in Europe— impact of cost considerations. Leukemia. 2007;21:383-6).

In the fifth and sixth aspects, an elevated level of BMI-I mRNA or protein is indicative that the patient may be suitable to receive an allogeneic stem cell transplant.

As indicated above in relation to the first and second aspects, the level of BMI-I mRNA or protein that is indicative of a particular outcome may vary depending on the type of patient. The level may be determined by comparing levels in CML patients who fare well after transplant and those who have a poor outcome. The inventors have found that a level of BMI-I mRNA of more than the median for a cohort of patients at diagnosis of CML in the chronic phase is indicative of a good outcome following allogeneic SCT, as described in the Examples. It may be appropriate to set a different threshold level of BMI-I mRNA or protein to aid in categorising patient groups according to suitability to receive allogeneic SCT. For example, a level of BMI-I mRNA or protein that is greater than one, or greater than two standard deviations (SD), above the mean or median level of BMI-I mRNA in a population of CML patients who at diagnosis may have other poor risk factors is indicative of a good outcome. A level of BMI-I mRNA or protein that is at the highest quartile (upper quartile) in a population of CML patients at diagnosis may be indicative of a good outcome following allogeneic SCT. A level of BMI-I mRNA or protein that is below the first quartile (lower quartile) in a population of CML patients at diagnosis may be indicative of a poor outcome following allogeneic SCT, with higher incidence of acute graft- versus- host disease. The availability of patient samples collected at diagnosis of CML, and preserved, for example by cyropreservation, allows the level of BMI-I to be correlated with outcome retrospectively. Such analyses, conducted on a large group of patients, will allow the correlation between BMI mRNA or protein level and suitability to receive an allogeneic SCT to be refined.

It will be appreciated that not all patients are diagnosed with CML in chronic phase. The method is most suitable for assessing the suitability of chronic phase CML patients to receive an allogeneic SCT.

In the context of the fifth and sixth aspects of the invention, a "good outcome" may be reflected by a relatively long overall survival compared to a "poor outcome". Where the prognosis is for a "good outcome", the probability of transplant related mortality is typically lower than in the case of a "poor outcome". Typically, acute graft versus host disease, which is one of the major causes of transplant related mortality, is lower in occurrence and/or severity where the prognosis is for a "good outcome" rather than a "poor outcome".

With reference to the sixth aspect, methods of performing allogeneic SCT are known in the art. Typically high-dose chemotherapy and/or radiotherapy is performed as a "preparative" or "conditioning" regimen prior to allogeneic SCT. In addition to the "conditioning" regimen, the patient will typically receive immediately before and after the graft infusion a treatment for prophylaxis of graft-versus-host disease including different immunosuppressive drugs. Suitable prophylaxes are described in: Storb et al. Methotrexate and cyclosporine versus cyclosporine alone for prophylaxis of graft-versus-host disease in patients given HLA-identical marrow grafts for leukemia: long-term follow-up of a controlled trial. Blood. 1989;73: 1729-34, which describes that prophylaxis with methotrexate and cyclosporine allows improved early survival compared to prophylaxis with cyclosporine alone. Further suitable prophylaxes are reviewed in Goker et al (2001) Exp Hematol 29: 259-277.

Where a patient is not considered suitable to receive an allogeneic SCT, an alternative therapy is provided according to the sixth aspect of the invention. This may be any suitable therapy with reference to the individual patient. Preferably, the alternative therapy comprises or consists of administration of a tyrosine kinase inhibitor, preferably imatinib mesylate (Deininger M et al, The development of imatinib as a therapeutic agent for chronic myeloid leukemia. Blood. 2005;105:2640-53). If the patient is intolerant or unresponsive to imatinib, alternative tyrosine kinase inhibitors may be administered, such as nilotinib (Kantarjian H, Giles F, Wunderle L et al. Nilotinib in imatinib-resistant CML and Philadelphia chromosome-positive ALL. N Engl J Med. 2006;354:2542-2551) or dasatinib (Hochhaus et al., Dasatinib induces notable hematologic and cytogenetic responses in chronic-phase chronic myeloid leukemia after failure of imatinib therapy. Blood. 2007;109:2303-9). Other suitable drugs include antimetabolites such as cytarabine or hydroxyurea, alkylating agents such as busulfan, interferon alfa, homoharringtonine, or new generation tyrosine kinase inhibitors (Guilhot et al. Interferon alfa-2b combined with cytarabine versus interferon alone in chronic myelogenous leukemia. French Chronic Myeloid Leukemia Study Group. N Engl J Med. 1997;337:223-9; Quintas-Cardama et al., Phase I/II study of subcutaneous homoharringtonine in patients with chronic myeloid leukemia who have failed prior therapy. Cancer. 2007;l 09:248-55; Giles et al. MK-0457, a novel kinase inhibitor, is active in patients with chronic myeloid leukemia or acute lymphocytic leukemia with the T315I BCR-ABL mutation. Blood. 2007;109:500-2) .

A seventh aspect of the invention provides a method of aiding in assessing whether a patient selected to be transplanted with an allogeneic stem cell transplant for treatment of chronic myeloid leukemia should receive enhanced prophylaxis for graft versus host disease prior to being transplanted with the transplant, the method comprising providing a sample from the patient, determining the level of BMI-I mRNA or protein in the sample and assessing whether the level is indicative that enhanced prophylaxis for graft versus host disease may be beneficial.

An eighth aspect of the invention provides a method of treating a patient with chronic myeloid leukemia comprising: (i) providing a sample from the patient;

(ii) determining the level of BMI-I mRNA or protein in the sample;

(iii) if the level of BMI-I mRNA or protein in the sample is indicative that the patient may benefit from enhanced prophylaxis for graft versus host disease prior to transplantation with an allogeneic stem cell transplant, providing said enhanced prophylaxis; and

(iv) transplanting the patient with an allogeneic stem cell transplant.

With reference to the seventh and eighth aspects, suitable samples from a patient and methods of determining the level of BMI-I mRNA or protein are as described in relation to the preceding aspects. Methods of performing graft versus host disease prophylaxis for allogeneic SCT are known in the art, and are as described in relation to the sixth aspect. Acute graft versus host disease is a major cause of transplant related mortality in CML patient who receive allogeneic SCT (Sullivan et al, Influence of acute and chronic graft-versus-host disease on relapse and survival after bone marrow transplantation from HLA- identical siblings as treatment of acute and chronic leukemia. Blood. 1989;73: 1720-8). In the context of the seventh and eighth aspects of the invention, "enhanced prophylaxis for graft versus host disease" refers to a prophylaxis regimen which includes procedures or treatments that would not ordinarily be given to a patient receiving prophylaxis for graft versus host disease in the context of allogeneic SCT for treatment of CML, and particularly to those patients with a high level of BMI-I mRNA or protein. For example, enhanced prophylaxis may involve different procedures or treatments, and/or it may involve the use of known procedures or drugs, but for longer duration or at higher dosage.

Suitably the enhanced prophylaxis for graft versus host disease comprises T cell depletion. T cells may be depleted in the graft, for example by treating it ex vivo, such as with physical separation techniques, selective depletion with lectins, treatment with cytotoxic drugs or use of anti-T cell serum or monoclonal antibodies (Goker et al (2001) Exp Hematol 29: 259-277). Alternatively, T cells may be depleted in the recipient of the graft prior to transplantation. In certain cases, T cells may be depleted both in the graft and in the recipient. Antithymocyte or antilymphocyte globulins may be used for these purposes. (Mohty M. Mechanisms of action of antithymocyte globulin: T-cell depletion and beyond. Leukemia. 2007;21 :1387-94; Bacigalupo A. Antilymphocyte/thymocyte globulin for graft versus host disease prophylaxis: efficacy and side effects. Bone Marrow Transplant. 2005;35:225-31). Enhanced prophylaxis may include the administration of immunosuppressive drugs in addition to those typically administered to CML patients treated by allogeneic SCT. It will be appreciated that the probability of a patient having a "good outcome" after allogeneic SCT is improved where both the probability of transplant related mortality is low and the probability of disease relapse is low. In general, enhanced prophylaxis is not provided to all CML patients who receive allogeneic SCT as there may be undesired side effects associated with enhanced prophylaxis. In particular, there may be a higher risk of disease relapse. Higher rates of disease relapse have been found to occur where CML patients received T cell depletion as a prophylaxis for acute graft versus host disease (Goldman JM et al., Bone marrow transplantation for chronic myelogenous leukemia in chronic phase. Increased risk for relapse associated with T-cell depletion. Ann Intern Med. 1988;108(6):806-14). Where immune deficiency is prolonged, such as after enhanced prophylaxis, there may also be an increased risk of infectious complication (Bacigalupo (2005) supra).

Suitably, enhanced prophylaxis is applied where the level of BMI-I mRNA or protein is low but the decision is still made to perform an allogeneic SCT. For example, a level of BMI-I mRNA or protein of lower than the median for a cohort of patients at diagnosis of chronic myeloid leukemia may be indicative that the patient may benefit from enhanced prophylaxis for graft versus host disease prior to transplantation with an allogeneic stem cell transplant.

A ninth aspect of the invention provides a use of a reagent which selectively identifies BMI-I mRNA or protein in the assessment of the suitability to receive an allogeneic stem cell transplant of a patient having chronic myeloid leukemia, or the assessment of whether a patient selected to be transplanted with an allogeneic stem cell transplant for treatment of chronic myeloid leukemia should receive prophylaxis for graft versus host disease prior to being transplanted with the transplant. The reagent, including its preferred features, is as described above in relation to the second aspect. The reagent is suitable for use according to the methods of the seventh and eighth aspects. Preferably, the reagent is one or more oligonucleotides. Preferably, it is used in a Q-RT/PCR.

A tenth aspect of the invention provides a use of a reagent which selectively identifies BMI-I mRNA or protein in the manufacture of a composition for assessing the suitability to receive an allogeneic stem cell transplant of a patient having chronic myeloid leukemia, or the assessment of whether a patient selected to be transplanted with an allogeneic stem cell transplant for treatment of chronic myeloid leukemia should receive prophylaxis for graft versus host disease prior to being transplanted with the transplant. The composition comprises a reagent as in the ninth aspect.

An eleventh aspect of the invention provides a method of improving the outcome of an allogeneic stem cell transplant in a patient, which patient is transplanted with the allogeneic stem cell transplant as a therapy for chronic myeloid leukemia, the method comprising administering an agent which is capable of enhancing the expression or function of BMI-I mRNA or protein in the patient.

The inventors have surprisingly discovered that the reduced expression of BMI-I is a risk factor for transplant related mortality, associated with higher incidence of acute graft versus host disease. Therefore, enhancement of the expression or function of BMI-I mRNA or protein may contribute to improving the outcome of the allogeneic SCT. The agent may be or comprise a polynucleotide encoding BMI-I or leading to amplification of the BMI-I coding sequence, or leading to upreguation of BMI-I, as will be well known to those skilled in the art. For example, administration of GCSF, for example after graft infusion, may lead to a beneficial upregulation of BMI-I. A twelfth aspect of the invention provides a method of improving the outcome of an allogeneic stem cell transplant in a patient who is ineligible for allogeneic stem cell transplant as a therapy for chronic myeloid leukemia, the method comprising administering an agent which is capable of reducing the expression or function of BMI-I mRNA or protein in the patient.

The inventors have discovered that the elevated expression of BMI-I is associated with aggressive disease and more rapid progression to blast crisis. It is considered that any patient with high BMI-I should receive an allogeneic stem cell transplant as (a) they have an innate increased risk of progression (b) their transplant outcome is better with less acute GVHD. For those patients that are not able to receive an allogeneic stem cell transplant, reduction of the expression or function of BMI-I mRNA or protein may be useful by contributing to reducing the risk of disease progression. For example, in the case of no allotransplant, one may use SiRNA directed against BMIl as a monotherapy or as part of a combination with other validated therapies.

The agent, including preferred embodiments thereof, is as described in relation to the fourth aspect of the invention. Preferably the agent consists of or comprises an antisense RNA or siRNA.

For the eleventh aspect of the invention, preferably the patient is administered the agent shortly prior to or at substantially the same time as being transplanted with the allogeneic stem cell transplant. Administration could continue during the first months and years after transplantation, and even indefinitely. The patient may be administered the agent as a prophylaxis for graft versus host disease, particularly acute graft versus host disease, shortly before transplantation, for example less than 3 weeks, 2 weeks, 1 week, 3 days, 2 days, a day, 12 hours; 6 hours, 3 hours, 2 hours or less than 1 hour before transplantation.

A thirteenth aspect of the invention provides a system comprising: (i) a reagent for determining the level of BMI-I mRNA or protein in a sample from a patient and; either,

(ii) a reagent for determining the level of BCR-ABL mRNA or protein in a sample from a patient; or (iii) a reagent for determining the level of a further marker in a sample from a patient, which further marker is indicative of a particular outcome for a patient with chronic myeloid leukemia.

Surprisingly, the level of BMI-I mRNA or protein in the PBMCs of a patient diagnosed with CML, particularly in chronic phase, is useful in aiding in the prognosis of the patient and assessing whether the patient is suitable to receive an allogeneic stem cell transplant, as described above. Accordingly, the system of the thirteenth aspect may be used in either or both of aiding prognosis and suitability to receive an allogeneic stem cell transplant. The reagent for determining the level of BMI-I mRNA or protein may be as defined in relation to the preceding aspects of the invention, and may be used in relation to a sample from the patient as previously described Where the system comprises a reagent for determining the level of BCR-ABL mRNA or protein in a sample from a patient, a suitable reagent is as described in Gabert et al. Leukemia. 2003;l 7:2318-57. It is routine to measure BCR-ABL in CML patients, for example, to monitor the presence and degree of disease remission after imatinib administration or stem cell transplantation. Typically, the level of BCR-ABL increases with disease progression.

Where the system of the thirteenth aspect comprises a reagent for determining the level of a further marker in a sample from a patient, which further marker is indicative of a particular outcome for a patient with CML, the reagent for determining the level of the further marker may be as described above in relation to the first aspect of the invention. Preferably the further marker is proteinase-3 mRNA or protein.

A fourteenth aspect of the invention provides a method for identifying an agent useful in increasing the life expectancy of a patient in the chronic phase of chronic myeloid leukemia, the method comprising the steps of a) determining whether a test compound is capable of suppressing production of, or activity of, BMI-I in a sample from a patient with chronic myeloid leukemia and b) selecting a compound which is capable of suppressing production of, or activity of, BMI-I in a patient with chronic myeloid leukemia or in a sample from such a patient.

A fifteenth aspect of the invention provides a method for identifying an agent useful in improving the outcome of an allogeneic stem cell transplant in a patient, which patient is transplanted with the allogeneic stem cell transplant as a therapy for chronic myeloid leukemia, the method comprising the steps of a) determining whether a test compound is capable of increasing production of, or activity of, BMI-I in a sample from a patient with chronic myeloid leukemia and b) selecting a compound which is capable of increasing production of, or activity of, BMI-I in a patient with chronic myeloid leukemia or in a sample from such a patient.

It will be appreciated that the invention of the fourteenth aspect provides a screening assay for use in trying to identify drugs which may be useful in suppressing production of, or activity of, BMI-I. Agents identified in the methods may themselves be useful as a drug or they may represent lead compounds for the design and synthesis of more efficacious compounds. By "agent", we include all chemical entities as described in relation to the fourth aspect of the invention.

It will likewise be appreciated that the invention of the fifteenth aspect provides a screening assay for use in trying to identify drugs which may be useful in increasing production of, or activity of, BMI-I . Agents identified in the methods may themselves be useful as a drug or they may represent lead compounds for the design and synthesis of more efficacious compounds.

The agent may be a drug-like compound or lead compound for the development of a drug-like compound. It will be appreciated that the said method may be useful as screening assays in the development of pharmaceutical compounds or drugs, as well known to those skilled in the art. The term "drug-like compound" is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate cellular membranes, but it will be appreciated that these features are not essential.

The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.

All documents cited in the patent specification are hereby incorporated herein by reference.

The invention will now be described in more detail by reference to the following non-limiting Examples and Figures wherein:

Figure 1 - BMI-I expression in CML as assessed by Q-RT7PCR. (A) BMI-I expression in CD34+ immunomagnetically selected hematopoietic progenitors from CML patients at diagnosis in chronic phase (CP) as compared to patients in more advanced disease stage (acceleration phase and blast crisis). The definition of CP and advanced phases (accelerated phase and blast crisis) was based on previously established criteria.³'¹¹'²⁵: CP <10% blasts, accelerated phase 10-30% blasts or <10% blasts with clonal evolution, and blast crisis >30% blasts. Bone marrow (BM)-derived CD34+ cells from healthy donors, and G-CSF-mobilized CD34+ stem cells (PBSC) from non-CML donors were used as controls. As previously described, G-CSF-mobilized CD34+ PBSCs express high levels of BMI-I as compared to non-stimulated normal cells.¹⁰ (B) BMI-J expression in total unfractionated PBMCs from CML patients at diagnosis in CP as compared to patients in more advanced disease stage (accelerated phase and blast crisis). Total PBMCs from healthy donors were used as controls. (C) E2F-1 expression in total unfractionated PBMCs from CML patients at diagnosis in CP as compared to patients in more advanced disease stage (accelerated phase and blast crisis). Total PBMCs from healthy donors were used as controls. Horizontal bars denote the medians. Values of genes represent the Q-RT/PCR expression as a ratio of the gene of interest to the GAPDH control gene. For establishment of the Q-RT/PCR assay, the Jurkat and HeLa cell lines were used as a positive controls for BMI-I and E2F-1 expression, respectively, with a standard curve being generated for the amplification of logarithmic dilutions (10^"1 to 10^~5) of their cDNAs. An average of the duplicates of each datapoint was taken and plotted against the cycle threshold (Ct). The technical variability between duplicate samples in our RT/PCR assays has been established for a number of different genes as < 1.3-fold at the 95% level of confidence (data not shown).

Figure 2 - BMI-I expression and probabilities of overall survival. (A) BMI-I expression in CD34+ immunomagnetically selected hematopoietic progenitors from diagnosis in a cohort of 64 CP CML patients, showing different disease evolution patterns: patients who developed blast crisis (BC) within 3 years of diagnosis were defined as having "aggressive disease" (n=17), whereas those who survived for over 7 years prior to the onset of BC were defined as having "indolent disease" (n=23). Patients who survived between 3 and 7 years without developing BC were categorized as having "intermediate disease" (n=24). There was a significant difference in BMI-I expression among the 3 groups (P= 0.01 when comparing all three groups), between patients with "intermediate" and "aggressive" disease (P=O-Ol) and between "indolent" and "aggressive" disease (P= 0.01), but not between "indolent" versus "intermediate" disease, P=NS). The median age at diagnosis of the selected patients was 45.7 years (range 17.6-68.3). The male: female ratio was 1.8: 1 (41 males, 23 females). The majority of patients were diagnosed in the pre-imatinib era. (B) Overall survival according to BMI-I expression as assessed by Q-RT/PCR in the whole cohort of the above 64 patients. The median gene expression level is used to segregate the patients into a "low BMI-P' group (BMI-I expression<median) and a "high BMI-I" group (BMI-I expression>median). (C) Cox multivariate analysis yielded a model with the combination of low BMI-I and high proteinase-3 (PR-3) expression as predictive of significantly improved survival. The median gene expression levels were used to segregate the patients into a "low BMI-I- high PR-3" group (BMI-I expression<median and Pi?-3>median; n=21) and a "high BMI-I- low PR-3" group (BMI-I expression>median and Pi?-J<median; n=43). Values of genes represent the Q-RT/PCR expression as a ratio of the gene of interest to the GAPDH control gene.

Figure 3 - BMI-I expression and outcome. (A) Overall survival (OS) according to BMI-I expression as assessed by Q-RT/PCR in the whole cohort of 84 patients. (B) Leukemia-free survival (LFS) according to BMI-I expression. (C) Cumulative incidence of grade 2-4 acute GVHD according to BMI-I expression. In multivariate analysis, when comparing grade 0-2 or grade 2 acute GVHD versus the severe acute GVHD group (grade 3-4), the statistical association with BMI-I remains significant (P=0.01 and P=0.03, respectively). In terms of chronic GVHD, 74 patients survived to day 100 and were evaluable for chronic GVHD: 41 did not develop any form of chronic GVHD and 33 had a limited (n=4) or an extensive (n=29) form. In univariate analysis, no statistically significant associations were found between the four analyzed genes and chronic GVHD (BM-I, P=0.07; CD7, P=O.12; ELA-2, P=0.93; PR3, P=O.12). When added to a multivariate analysis, none of these genes was found to be significant. The median gene expression level is used to segregate the patients into a "low BMI-I" group (BMI-I expression <median) and a "high BMI-I" group (BMI-I expression >median). Values of BMI-I represent the Q- RT/PCR expression as a ratio to the GAPDH control gene. For establishment of the Q-RT/PCR assay, the Jurkat cell line was used as a positive control for BMI-I expression with a standard curve being produced for the amplification of logarithmic dilutions (10-1 to 10-5) of its cDNA. An average of the duplicates of each datapoint was taken and plotted against the cycle threshold (Q). The technical variability between duplicate samples in our RT/PCR assays has been established for a number of different genes as < 1.3-fold at the 95% level of confidence.

Example 1: The polycomb group BMI-I gene is a molecular marker for predicting prognosis of chronic myeloid leukemia

Abstract

Because the polycomb group gene BMI-I regulates the proliferation of both normal and leukemic stem cells, we examined whether BMI-I expression was associated with disease progression in chronic myeloid leukemia (CML). Levels of BMI-I RNA were significantly higher in patients with advanced phase than in patients with chronic phase CML in both CD34+ cells (P=0.006) and total peripheral blood mononuclear cells (PO.0001). E2F-1, a transcription factor regulating BMI-I, was upregulated in CML as compared to controls (P=O-OOl).

In a cohort of 64 CML patients, the level of BMI-I at diagnosis correlated with time to transformation to blast crisis, and the combination of low BMI-I and high proteinase-3 expression was associated in multivariate analysis with an improved overall survival (P=O.001). We conclude that BMI-I may be a biomarker for the intrinsic heterogeneity of CML, and its measurement at diagnosis can help predict overall survival and thus contribute to better therapeutic decisions.

Introduction

Despite a consistent molecular abnormality, the BCR-ABL oncogene, chronic myeloid leukemia (CML) exhibits marked heterogeneity in prognosis, and various attempts have been made to determine prognosis for individual patients at the time of diagnosis in chronic phase (CP).¹ For example, the Sokal and Hasford prognostic scores derived from study of patients treated predominantly with busulfan and interferon-α, respectively, have proved moderately useful in predicting the duration of survival for individual patients², and recent clinical experience suggests that this heterogeneity is still reflected in response to therapy with imatinib³.

The polycomb group (PcG) gene BMI-I plays an essential role in regulating the proliferative activity of both normal and leukemic stem cells.⁴'⁵ BMI-I is a transcriptional repressor likely restricted to stem cells and progenitors.

Coexpression of BMI-I and other proteins from the PcG, such as EZH2, confers a higher degree of malignancy.⁶ BMI-I overexpression was described in several types of cancer, including hematological neoplasms.^7"10 We therefore measured BMI-I expression in CML to discover whether it might be involved in pathogenesis, and might serve as a biomarker to predict disease aggressiveness and progression from CP to more advanced phases.

Materials and methods

Patients and controls

Two independent cohorts of CML patients were studied: 1) patients in CP whose nucleated cells were collected by leukapheresis and cryopreserved within 3 months of diagnosis, before start of treatment, and for whom complete follow-up was available (n=64)^u; 2) patients with cryopreserved cells collected at CP or blast crisis (BC). Informed consent for the use of these cells for research was obtained according to the requirements of the Local Research Ethics Committee. Diagnosis of CML and disease staging was based on clinical parameters and morphology of blood and bone marrow.¹¹ Peripheral blood mononuclear cells (PBMC) from healthy donors, granulocyte-colony-stimulating factor (G-CSF)- mobilized peripheral blood stem cells (PBSC) from non-CML patients, and bone marrow from healthy donors (StemCell, London, UK) were also obtained by informed consent and were used as controls.

Quantitative real-time reverse transcription and polymerase chain reaction (Q- RT /P CR) amplification

PBMCs from cryopreserved material were isolated by density gradient centrifugation (Lymphoprep, Nycomed, Oslo, Norway). CD34+ cells were selected by binding to immunomagnetic beads (MiniMACS, Miltenyi Biotech, Bergisch-Gerbach, Germany). Total RNA was extracted using the Qiagen RNeasy kit (Qiagen, Crawley, UK), treated with DNase I (Invitrogen, Paisley, UK) to eliminate genomic DNA, and reverse-transcribed into cDNA according to standard methods. Expression of BMI-I, E2F-1 and GAPDH was assessed by Q- RT/PCR using the Applied Biosystems 7300/7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA). All Q-RT/PCR reactions were performed in 25-μL volume." GAPDH expression was used as the endogenous cDNA quality control. The ABI Assays-on-demand™ TaqMan™ probe-and- primer reagents for BMI-I, E2F-1, and GAPDH were utilized according to the manufacturer's instructions.

Statistical methods

Groups were compared using the Mann- Whitney test for continuous data and Fisher's exact test for categorical data. Survival curves were calculated using the

Kaplan-Meier method, and groups compared using the log-rank test. Patients were divided into groups using Q-RT/PCR values delineated by the median.

Genes or parameters identified from the univariate analysis with p-values of less than 0.20 (PO.20) were entered into a Cox regression analysis, and a forward and backward stepping procedure was used to find the best model to predict survival. All quoted P-values are from two-sided tests with values <0.05 considered significant.

Results and Discussion

BMI-I expression levels in CD34+ cells were significantly lower in CP (n=13) than in more advanced stages (accelerated phase and BC, n=17) of CML (P=O.006; Figure IA). The same significant difference held also true when BMI-I expression was compared in unfractionated CML-derived PBMCs (PO.0001; Figure IB). In 8 patients for whom both CD34+ cells and PBMCs were available, a paired comparison disclosed a trend towards a significant correlation between BMI-I expression in the two cell populations (P=O.05; Pearson correlation R=O.7). Of note, BMI-I expression in bone marrow-derived CD34+ stem cells from healthy donors was significantly lower as compared to CML patients (P=O.003; Figure IA). In order to gain insights into the mechanisms underlying BMI-I upregulation in CML, we also assessed the expression of E2F-1, a transcription factor that controls various genetic programs including cell cycle progression and apoptosis,¹² and that was shown to directly regulate BMI-I activity.¹³ We found that PBMCs from CML patients (all disease stages) displayed significantly higher levels of E2F-1 as compared to healthy controls (P^O.OOl; Figure 1C).

We have previously shown that the combination of CDl, proteinase-3 (PR-3) and elastase-2 (ELA-2) expression levels at diagnosis can reflect the intrinsic molecular heterogeneity of CML in CP, especially duration of CP. This was observed in patients with an "aggressive disease" who develop BC early after diagnosis (<3 years), as opposed to patients with an "indolent disease" whose BC occurs >7 years after diagnosis.¹¹ We therefore measured BMI-I expression in CD34+ cells from a cohort of 64 CP CML patients. Characteristics of the patients are shown in Table 1.

Table 1 : Patient characteristics(*) and prognostic features

(*) The majority of these patients was from the pre-imatinib era and had received more than one type of treatment over the course of their disease, including busulfan, hydroxyurea, interferon-alpha, autologous or allogeneic stem cell transplantation.

We found a significant difference in BMI-I levels between patients with an "indolent" or an "intermediate" (patients surviving between 3 and 7 years without developing BC) clinical pattern as compared to those who had an "aggressive" clinical evolution (P=0.01 for comparing the 3 groups, Kruskal-Wallis test; Figure 2A). Patients displaying a low BMI-I expression level at diagnosis had significantly longer survival than other patients. (P=O-OOS; Figure 2B). When BMI-I was included in a Cox multivariate survival analysis model (together with the previously established prognostic markers, CDl, ELA-2, PR-3, and other relevant demographic and clinical parameters as indicated in Table 1 above), the combination of low BMI-I and high PR-3 expression levels was found to be a strong independent marker associated with significantly longer overall survival (P=O-OOl; RR=0.20, 95%CI; 0.08-0.54; Figure 2C).

Our observations suggest an important role for BMI-I in CML pathophysiology and prognosis. BMI-I is essential for the self-renewal of both hematopoietic and neuronal stem cells, as well as cancer stem cells.⁵'¹⁴'¹⁵ It has also been shown to cooperate with MYC in the generation of lymphomas in double transgenic mice.¹⁶ Furthermore, BMI-I blocks senescence and immortalizes mouse embryo fibroblasts and, in combination with an activated H-RAS gene, leads to neoplastic transformation.¹⁷ These oncogenic functions depend in part on the ability of BMI- 1 to repress the INK4A locus, which encodes the tumor suppressor proteins pl6^Ink4a and pi 4^V⁸ All these pathways are known to be involved in the proliferation of BCR-ABL positive cells,¹⁹ suggesting that overexpression of BMI- 1 acts in conjunction with its related partner genes in the genesis and transformation of CML, in a manner analogous to its role in other malignancies.

Though our data do not provide a complete picture of the mechanisms involved in BMI-I upregulation in CML, they show that these likely involve the E2F-1 gene, which we also found to be overexpressed in CML. Thus, E2F-1 (i) directly regulates BMI-I,¹³ (H) has its activity controlled by the retinoblastoma-cyclin pathway²⁰ and, (Hi) via this pathway, defines a route from Bcr-Abl to MYC transcription, which is required for Bcr-Abl transformation²¹. This implies that genetic alterations impairing E2F-1, BMI-J and their downstream targets may render hematopoietic cells refractory to the induction of differentiation, as previously demonstrated in myeloid cell line models, and are thereby likely to play a major role in the progression and aggressiveness of CML. Moreover, induction of BMI-I would change the composition of the PcG complex to favor proliferation over cell cycle arrest, since the relative amounts of BMI-I in the complex determine its biochemical and biological functions.²³ The identification of 5M7-7 -cooperative factors in CML will surely help defining it as a bonafide cancer stem cell inducer.

From the clinical standpoint, our findings demonstrate that BMI-I can serve as a novel molecular marker to predict prognosis in CML, particularly in conjunction with the expression level of immune-related proteins such as PR-3.¹¹'²⁴ An interesting and useful aspect of our study, from a practical point of view, was the indication that the expression of BMI-I in CD34+ cells tends to parallel that found in total PBMCs, as these provide a more easily obtainable and less expensive biological material in which a rapid Q-RT/PCR prognostication test can be done at diagnosis of the disease. Despite their great success, it is still unclear whether tyrosine kinase inhibitors can cure CML. Therefore, the prospective screening for BMI-I expression in combination with other molecular markers,²⁵ can help refining CML disease staging and prognosis towards optimizing therapeutic interventions, including perhaps BMI- 1 -targeted inhibitors.

References

1. O'Brien SG, Guilhot F, Larson RA, et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med. 2003;348:994-1004.

2. White D, Saunders V, Lyons AB, et al. In vitro sensitivity to imatinib- induced inhibition of ABL kinase activity is predictive of molecular response in patients with de novo CML. Blood. 2005; 106:2520-2526. 3. Hughes TP, Kaeda J, Branford S, et al. Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med. 2003;349:1423-1432.

4. Jacobs JJ, Scheijen B, Voncken JW, Kieboom K, Berns A, van Lohuizen M. Bmi-1 collaborates with c-Myc in tumorigenesis by inhibiting c-Myc-induced apoptosis via INK4a/ARF. Genes Dev. 1999; 13:2678-2690.

5. Lessard J, Sauvageau G. Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature. 2003;423:255-260.

6. Cao R, Tsukada Y, Zhang Y. Role of Bmi-1 and RinglA in H2A ubiquitylation and Hox gene silencing. MoI Cell. 2005;20:845-854.

7. Bea S, Tort F, Pinyol M, et al. BMI-1 gene amplification and overexpression in hematological malignancies occur mainly in mantle cell lymphomas. Cancer Res. 2001 ;61 :2409-2412.

8. Kim JH, Yoon SY, Kim CN, et al. The Bmi-1 oncoprotein is overexpressed in human colorectal cancer and correlates with the reduced pl6INK4a/pl4ARF proteins. Cancer Lett. 2004;203:217-224.

9. Vonlanthen S, Heighway J, Altermatt HJ, et al. The bmi-1 oncoprotein is differentially expressed in non-small cell lung cancer and correlates with INK4A- ARF locus expression. Br J Cancer. 2001;84:1372-1376. 10. Mihara K, Chowdhury M, Nakaju N, et al. Bmi-1 is useful as a novel molecular marker for predicting progression of myelodysplastic syndrome and patient prognosis. Blood. 2006; 107:305-308.

11. Yong AS, Szydlo RM, Goldman JM, Apperley JF, MeIo JV. Molecular profiling of CD34+ cells identifies low expression of CD7, along with high expression of proteinase 3 or elastase, as predictors of longer survival in patients with CML. Blood. 2006;107:205-212.

12. Bracken AP, Ciro M, Cocito A, Helin K. E2F target genes: unraveling the biology. Trends Biochem ScL 2004;29:409-417.

13. Nowak K, Kerl K, Fehr D, et al. BMI-1 is a target gene of E2F-1 and is strongly expressed in primary neuroblastomas. Nucleic Acids Res. 2006;34: 1745-

1754. 14. Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke MF, Morrison SJ. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature. 2003;425:962-967.

15. Park IK, Qian D, Kiel M, et al. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature. 2003;423:302-305.

16. van Lohuizen M, Verbeek S, Scheijen B, Wientjens E, van der Gulden H, Berns A. Identification of cooperating oncogenes in E mu-myc transgenic mice by provirus tagging. Cell. 1991 ;65:737-752.

17. Jacobs JJ, Kieboom K, Marino S, DePinho RA, van Lohuizen M. The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature. 1999;397: 164- 168.

18. Bruggeman SW, Valk-Lingbeek ME, van der Stoop PP, et al. Ink4a and Arf differentially affect cell proliferation and neural stem cell self-renewal in BMI-1 -deficient mice. Genes Dev. 2005;19:1438-1443. 19. Goldman JM, MeIo JV. Chronic myeloid leukemia—advances in biology and new approaches to treatment. N Engl J Med. 2003;349:1451-1464. 20. Hernando E, Nahle Z, Juan G, et al. Rb inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control. Nature. 2004;430:797-802. 21. Stewart MJ, Litz- Jackson S, Burgess GS, Williamson EA, Leibowitz DS, Boswell HS. Role for E2F1 in p210 BCR-ABL downstream regulation of c-myc transcription initiation. Studies in murine myeloid cells. Leukemia. 1995;9:1499- 1507.

22. Amanullah A, Hoffman B, Liebermann DA. Deregulated E2F-1 blocks terminal differentiation and loss of leukemogenicity of Ml myeloblasts leukemia cells without abrogating induction of pl5(INK4B) and pl6(INK4A). Blood. 2000;96:475-482.

23. Dahiya A, Wong S, Gonzalo S, Gavin M, Dean DC. Linking the Rb and polycomb pathways. MoI Cell. 2001;8:557-569. 24. Molldrem JJ, Lee PP, Wang C, et al. Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia. Nat Med. 2000;6: 1018-1023. 25. Radich JP, Dai H, Mao M, et al. Gene expression changes associated with progression and response in chronic myeloid leukemia. Proc Natl Acad Sci U S A. 2006; 103:2794-2799.

Example 2 — Association between BMI-I expression, acute graft-versus-host disease and outcome following allogeneic stem cell transplantation in chronic myeloid leukemia

Abstract

Expression of CD7, ELA-2, PR-3 and the polycomb group gene BMI-I reflects the intrinsic heterogeneity and predicts prognosis of chronic myeloid leukemia (CML) patients who were not treated with allogeneic stem cell transplantation (allo-SCT). This study investigated whether expression of these genes determined outcome following allo-SCT in a cohort of 84 chronic phase (CP) CML patients. We found that patients expressing BMI-I at 'high' level before allo-SCT had an improved overall survival (P=O.005) related to a reduced transplant-related mortality. In multivariate analysis, when adjusted for the EBMT-Gratwohl score and other prognostic factors, there was an independent association between BMI- 1 expression and grade 2-4 acute graft-versus-host disease (RR=2.85, 95%CI; 1.3-6.4; P=0.011) suggesting that BMI-I measured prior to allo-SCT can serve as a biomarker for predicting outcome in CP-CML patients receiving allo-SCT, and may thus contribute to better therapeutic decisions.

Introduction

The management of chronic myeloid leukemia (CML) has changed radically since the introduction of tyrosine-kinase inhibitors. The decision whether to offer a patient allogeneic stem cell transplantation (allo-SCT) must be carefully weighed. Historically, various attempts have been made to determine prognosis and outcome for individual CML patients at the time of allo-SCT. Thus the EBMT-Gratwohl score based on histocompatibility, stage of disease at time of allo-SCT, age and sex of donor and recipient, and time from diagnosis to allo- SCT, proved to be the most useful scoring system to assess the risk of mortality when counseling and decision-making.¹'² However, it is established that CML exhibits marked heterogeneity in prognosis,³ that is reflected even in response to modern therapies.⁴ We have recently shown in CML patients who did not receive allo-SCT that the combination of CD7, proteinase-3 (PR-3), elastase-2 (ELA-2) and the polycomb group (PcG) gene BMI-I expression levels at diagnosis can reflect the intrinsic molecular heterogeneity of the disease, especially disease aggressiveness and duration of chronic phase (CP).⁵'⁶ As immune responses to both PR-3 and ELA-2 peptides have been implicated in the eradication of CML following allo-SCT,⁷'⁸ we investigated whether the above genes might also serve as bio markers to predict outcome of CP-CML patients receiving allo-SCT.

Materials and methods

Patients

A cohort of 84 CP-CML patients whose nucleated cells were collected by leukapheresis and cryopreserved prior to allo-SCT, and for whom complete follow-up was available, was analyzed. Informed consent for the use of these cells for research was obtained with approval from the Hammersmith and Queen Charlotte's and Chelsea Research Ethics Committee Institutional Review Board. Diagnosis of CML and disease staging was based on clinical parameters and morphology of blood and bone marrow.⁵'⁹ Acute and chronic graft- versus-host disease (GVHD) were graded according to classical criteria.¹⁰'¹¹

Quantitative real-time reverse transcription and polymerase chain reaction (Q- RT/PCR) amplification

PBMCs from cryopreserved material were isolated by density gradient centrifugation (Lymphoprep, Nycomed, Oslo, Norway). Total RNA was extracted using the Qiagen RNeasy kit (Qiagen, Crawley, UK), treated with DNase I (Invitrogen, Paisley, UK) to eliminate genomic DNA, and reverse- transcribed into cDNA according to standard methods. Expression of CD7, PR-3, ELA-2, BMI-I and GAPDH was assessed by Q-RT/PCR using the Applied Biosystems 7300/7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA). AU Q-RTVPCR reactions were performed in 25-μL volume.⁵ GAPDH expression was used as the endogenous cDNA quality control. The ABI Assays-on-demand™ TaqMan™ probe-and-primer reagents for CD7, PR-3, ELA-2, BMI-I, and GAPDH were utilized according to the manufacturer's instructions. The median gene expression level was used to segregate the patients into a "low" group (gene expression <median) and a "high" group (gene expression >median).

Statistical methods The following post allo-SCT outcome parameters were analyzed: engraftment, acute GVHD, chronic GVHD, transplant-related mortality (TRM), leukemia-free- survival (LFS) and overall survival (OS). Groups were compared using the Mann- Whitney test for continuous data and Fisher's exact test for categorical data. Probabilities of OS and LFS were calculated using the Kaplan-Meier method, and groups compared using the log-rank test. The cumulative incidence procedure was used to calculate probabilities of TRM and GVHD. Genes identified from univariate analyses with P-values of less than 0.20 (PO.20) were entered into Cox regression analyses that contained established prognostic variables (Gratwohl Score, year of transplant, GVHD prophylaxis regimen). All quoted P-values are from two-sided tests with values <0.05 considered significant.

Results and Discussion

Patient, disease and transplant characteristics are summarized in Table 1. Briefly, all patients received allo-SCT from an HLA-identical sibling. Patients were mainly treated in the pre-imatinib era. The median follow-up time for the patients alive post-allo-SCT was 9.8 (range 1.7 - 23.9) years. Patients were transplanted at a median of 9 months from diagnosis (range, 3- 94). The median EBMT- Gratwohl calculated score was 3. In this series, 20 patients (24%) died of TRM, while 6 patients died of disease progression. CD7, PR-3, ELA-2 and BMI-I expression was assessed by Q-RT/PCR in recipient PBMCs in the whole cohort of 84 patients. The median expression level for each individual gene was used to segregate the patients into 2 groups ("low": gene expression <median, and "high": gene expression >median). None of the 4 tested genes showed any significant association with neutrophil or platelet engraftment, or with graft rejection. CD7, PR-3 and ELA-2 expression was not associated with OS. However, and in sharp contrast to our previous findings in the non-allo-SCT setting,⁶ patients displaying a "high" BMI-I expression level prior to allo-SCT had significantly better OS than those with low expression (P=O.005; Figure 3A). Furthermore, when BMI-I expression was included in a multivariate survival model and adjusted for the other prognostic variables (EBMT-Gratwohl score, allo-SCT era, and other relevant demographic and transplant-related parameters detailed in Table 2), a high expression level was found to be an independent marker associated with better survival (RR=2.72, 95%CI; 1.1-6.9; P=0.034). In this multivariate model, the predictive value of BMI-I expression in terms of OS was also significant when the analysis was restricted to patients (n=68) who did not receive any form of T-cell depletion for GVHD prophylaxis (RR=4.19, 95%CI; 1.31-13.4, P=O-Ol 5).

Table 2. Patient, disease and transplant characteristics.

Abbreviations: CML, chronic myeloid leukemia; allo-SCT, allogeneic stem cell transplantation; GVHD, graft-versus-host disease; CMV, cytomegalovirus. AU patients received allo-SCT from an HLA-identical sibling, with 81 (96%) receiving a bone marrow graft, while 3 patients (4%) received peripheral blood stem cells. The median expression level for BMI-I was used to segregate the patients into 2 groups ("low": gene expression < median, and "high": gene expression > median).

* Comparison between the "low" and "high" BMI-I groups.

Given the impact of BMI-I expression level on OS, without a significant association with relapse, and since neither BMI-I, nor the other tested genes showed any significant association with LFS (Figure 3B), we assessed their impact on TRM. In multivariate analysis, BMI-I and ELA-2 showed statistically significant associations with TRM (RR=3.02, 95%CI; 1.05-8.71 ; P=0.041 and RR=2.7, 95%CI; 1.02-7.16; P=0.045 respectively). Expression of CD7, PR-3 and ELA-2 was not associated with acute GVHD occurrence or severity. However, there was a striking and significant association between acute GVHD and BMI-I expression, not only in overall incidence ("low" BMI-I : grade 0-1 (n-21), grade 2 (n=10), grade 3-4 (n=9); "high" BMI-I : grade 0-1 (n=32), grade 2 (n=9), grade 3-4 (n=l); P=0.005) but also in cumulative incidence at 100 days (24% vs. 48%; P=O.016, Figure 3C). In multivariate analysis a "low" BMI-I expression level was associated with an increased risk of grade 2-4 acute GVHD (RR=2.85, 95%CI; 1.3-6.4; P=O-OI l).

Our previous observations suggested that BMI-I, an essential gene for the self- renewal of normal as well as cancer stem cells,^12"14 plays an important role in CML pathophysiology and prognosis in the non-transplant setting.⁶ However, it is remarkable that, whereas in patients treated with hydroxyurea and interferon-α (in the pre-imatinib era) high expression of BMI-I was associated with a worse prognosis, the opposite was observed in patients treated with allo-SCT. Several hypotheses can be offered to help understanding these new and somewhat unexpected findings in CML patients who receive allo-SCT. As previously established in CML and other malignancies,⁶'^15"18, BMI-I, acting in cooperation with other oncogenes, can induce neoplastic transformation,¹⁹'²⁰ and its overexpression contributes to disease aggressiveness. However, in the context of allo-SCT for CP-CML, a gain of a neoplastic proliferative advantage within the leukemia stem cell pool through increased BMI-I expression may be neutralized by an immune response in donor cells against BMI-I, which was shown to be a genuine tumor-associated antigen.²¹ Moreover, our data on the association between BMI-I and acute GVHD add to the growing evidence that PcG genes are also involved in the regulation of immune functions. For example, Hosokawa et al. demonstrated that higher expression of BMI-I facilitates Th2 cell differentiation in a Ring finger-dependent manner by regulating GAT A3 protein stability,²² reinforcing the view that the balance between ThI and Th2 immune responses is critical for controlling the severity of acute GVHD.²³'²⁴ Therefore, if validated in prospective independent series, BMI-I can represent a potential "bio marker" to identify those patients at higher risk of GVHD development.

Overall, our findings suggest that BMI-I, in addition to the well-established EBMT-Gratwohl score,¹ can improve risk assessment of candidate CML patients for allo-SCT. At present, long term results of tyrosine-kinase inhibitors are still under considerable debate, unlike allo-SCT for which evidence is solid and long- standing. Allo-SCT is a viable option for 25% of CML patients who exhibit primary or secondary resistance to tyrosine-kinase inhibitors, and remains the first-line therapy in many countries due to specific economical reasons.²⁵ However, the risk of high TRM associated with acute GVHD must be balanced against the benefit of allo-SCT, hence the current challenge to identify subsets of patients best suited to receive this type of therapy. In this regard, and in addition to other immune parameters,⁸ BMI-I is likely to represent a marker of CML outcome after allo-SCT, and may offer a valuable tool towards tailored therapeutic interventions, including informed recommendation for allo-SCT.

References

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2. Passweg JR, Walker I, Sobocinski KA, Klein JP, Horowitz MM, Giralt SA. Validation and extension of the EBMT Risk Score for patients with chronic myeloid leukaemia (CML) receiving allogeneic haematopoietic stem cell transplants. Br J Haematol. 2004;125:613-620. 3. Hughes TP, Kaeda J, Branford S, et al. Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med. 2003 ;349: 1423- 1432. 4. O'Brien SG, Guilhot F, Larson RA, et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med. 2003 ;348:994- 1004.

5. Yong AS, Szydlo RM, Goldman JM, Apperley JF, MeIo JV. Molecular profiling of CD34+ cells identifies low expression of CD7, along with high expression of proteinase 3 or elastase, as predictors of longer survival in patients with CML. Blood. 2006;l 07:205-212.

6. Mohty M, Yong AS, Szydlo RM, Apperley JF₅ MeIo JV. The polycomb group BMIl gene is a molecular marker for predicting prognosis of chronic myeloid leukemia. Blood. 2007,110:380-383.

7. Molldrem JJ, Lee PP, Wang C, et al. Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia. Nat Med. 2000;6:1018-1023.

8. Yong AS, Rezvani K, Savani BN, et al. High PR3 or ELA2 expression by CD34+ cells in advanced-phase chronic myeloid leukemia is associated with improved outcome following allogeneic stem cell transplantation and may improve PRl peptide-driven graft-versus-leukemia effects. Blood. 2007,110:770- 775.

9. Radich JP, Dai H, Mao M, et al. Gene expression changes associated with progression and response in chronic myeloid leukemia. Proc Natl Acad Sci U S

A. 2006; 103:2794-2799.

10. Gratwohl A, Hermans J, Apperley J, et al. Acute graft- versus-host disease: grade and outcome in patients with chronic myelogenous leukemia. Working Party Chronic Leukemia of the European Group for Blood and Marrow Transplantation. Blood. 1995,86:813-818.

11. Shulman HM, Sullivan KM, Weiden PL, et al. Chronic graft-versus-host syndrome in man. A long-term clinicopatho logic study of 20 Seattle patients. Am J Med. 1980;69:204-217.

12. MoIo fsky AV, Pardal R, Iwashita T, Park IK, Clarke MF, Morrison SJ. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature. 2003,425:962-967.

13. Lessard J, Sauvageau G. Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature. 2003;423:255-260.

14. Park IK, Qian D, Kiel M, et al. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature. 2003,423:302-305.

15. Bea S, Tort F, Pinyol M, et al. BMI-1 gene amplification and overexpression in hematological malignancies occur mainly in mantle cell lymphomas. Cancer Res. 2001 ;61 :2409-2412.

16. Mihara K, Chowdhury M, Nakaju N, et al. Bmi-1 is useful as a novel molecular marker for predicting progression of myelodysplastic syndrome and patient prognosis. Blood. 2006;l 07:305-308.

17. Kim JH, Yoon SY, Kim CN, et al. The Bmi-1 oncoprotein is overexpressed in human colorectal cancer and correlates with the reduced pl6INK4a/pl4ARF proteins. Cancer Lett. 2004;203:217-224. 18. Chowdhury M, Mihara K, Yasunaga S, Ohtaki M, Takihara Y, Kimura A. Expression of Po Iy comb-group (PcG) protein BMI-1 predicts prognosis in patients with acute myeloid leukemia. Leukemia. 2007,21:1116-1122. 19. van Lohuizen M, Verbeek S, Scheijen B, Wientjens E, van der Gulden H, Bems A. Identification of cooperating oncogenes in E mu-myc transgenic mice by provirus tagging. Cell. 1991 ;65:737-752.

20. Jacobs JJ, Kieboom K, Marino S, DePinho RA, van Lohuizen M. The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature. 1999;397: 164- 168.

21. Steele JC, Torr EE, Noakes KL, et al. The polycomb group proteins, BMI-I and EZH2, are tumour-associated antigens. Br J Cancer. 2006;95:1202- 1211. 22. Hosokawa H, Kimura MY, Shinnakasu R, et al. Regulation of Th2 cell development by Polycomb group gene bmi-1 through the stabilization of GATA3. J Immunol. 2006; 177:7656-7664.

23. Mohty M, Blaise D, Faucher C, et al. Inflammatory cytokines and acute graft- versus-ho st disease after reduced-intensity conditioning allogeneic stem cell transplantation. Blood. 2005;106:4407-4411.

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Example 3: Hypothetical treatment of a 54 year old CML patient

A 54 year old man might have been diagnosed with CML two years ago, have received imatinib and be developing resistance to imatinib. An HLA compatible donor might be available, but also other treatment approaches currently under investigation might be available, such as dasatinib, HHT or aurora kinase inhibitors. Such a patient would be likely to benefit from an allogeneic stem cell transplantation. However, because of his disease status, and advanced age (54 is old for stem cell transplantation), this patient would be at "high" risk for GVHD and therefore for a fatal toxicity. If his BMI-1 level is "high", a clinician would be more likely to perform a transplant than if his BMI-1 level is "low". If the patient, in consultation with the clinician, decided to proceed to transplantation, knowledge of the BMI-1 status could guide treatment approaches. If the BMI-1 level was "high", a standard GVHD prophylaxis regimen might be used, as this would potentially be of most benefit to the patient. If the patient is in a "low" BMI-1 category, an enhanced GVHD prophylaxis regimen might be used in order to minimize GVHD risk, although the risk of infection would be slightly higher and the risk of CML relapse might be increased.

Claims

1. A method of aiding in prognosing chronic myeloid leukemia in a patient who has chronic myeloid leukemia, the method comprising providing a sample from the patient, determining the level of BMI-I mRNA or protein in the sample and assessing whether the level is indicative of a particular outcome for the patient.

2. A method according to Claim 1 wherein an elevated level of BMI-I mRNA or protein is indicative of a poor outcome.

3. A method according to Claim 1 or 2 wherein the sample is a CD34⁺ cell sample or a peripheral blood mononuclear cell sample.

4. A method according to any preceding claim wherein the sample is collected from the patient at the time of diagnosis of chronic myeloid leukemia.

5. A method according to any preceding claim wherein the level of BMI-I mRNA is measured.

6. A method according to Claim 5 wherein the level of BMI-I mRNA is measured by quantitative real time PCR.

7. A method according to Claim 1 further comprising determining the levels of one or more further chronic myeloid leukemia markers in a sample from the patient and assessing whether the levels of said further marker or markers is indicative of a particular outcome for the patient.

8. A method according to Claim 7 wherein the level of BMI-I mRNA or protein and the level of the one or more chronic myeloid leukemia markers are all taken into account when assessing whether the levels are indicative of a particular outcome for the patient.

9. A method according to Claim 7 or 8 wherein the further marker is proteinase-3.

10. Use of a reagent which selectively identifies BMI-I mRNA or protein in the prognosis of chronic myeloid leukemia in a patient.

11. Use of a reagent which selectively identifies BMI-I mRNA or protein and use of a reagent that selectively identifies a further chronic myeloid leukemia marker in the prognosis of chronic myeloid leukemia in a patient.

12. Use according to any one of Claims 9 to 11 wherein the reagent is an oligonucleotide.

13. Use according to Claim 12 in a quantitative real time PCR.

14. Use of a reagent which selectively identifies BMI-I mRNA or protein in the manufacture of a composition for prognosing chronic myeloid leukemia in a patient.

15. A method of increasing the life expectancy of a patient in the chronic phase of chronic myeloid leukemia comprising administering a compound which is capable of reducing the expression or function of BMI-I mRNA or protein in the patient.

16. A method according to Claim 15 wherein the compound consists of or comprises an antisense RNA or siRNA.

17. A method of aiding in assessing a patient having chronic myeloid leukemia for suitability to receive an allogeneic stem cell transplant, the method comprising providing a sample from the patient prior to starting the conditioning or preparative regimen for transplantation, determining the level of BMI-I mRNA or protein in the sample and assessing whether the level is indicative of suitability to receive an allogeneic stem cell transplant.

18. A method of treating a patient with chronic myeloid leukemia comprising:

(i) providing a sample from the patient;

(ii) determining the level of BMI-I mRNA or protein in the sample;

(iii) assessing whether the level of BMI-I mRNA or protein in the sample is indicative of suitability to receive an allogeneic stem cell transplant; and,

(iv) if the level of BMI-I mRNA or protein in the sample is indicative that the patient may be suitable to receive an allogeneic stem cell transplant, performing an allogeneic stem cell transplant; and if the level of BMI-I mRNA or protein in the sample is not indicative that the patient may be suitable to receive an allogeneic stem cell transplant, treating the patient with an alternative therapy.

19. A method according to Claim 17 or 18 wherein an elevated level of BMI-I mRNA or protein is indicative that the patient may be suitable to receive an allogeneic stem cell transplant.

20. A method of aiding in assessing whether a patient selected to be transplanted with an allogeneic stem cell transplant for treatment of chronic myeloid leukemia should receive enhanced prophylaxis for graft versus host disease prior to being transplanted with the transplant, the method comprising providing a sample from the patient, determining the level of

BMI-I mRNA or protein in the sample and assessing whether the level is indicative that enhanced prophylaxis for graft versus host disease may be beneficial.

21. A method of treating a patient with chronic myeloid leukemia comprising: (i) providing a sample from the patient;

(ii) determining the level of BMI-I mRNA or protein in the sample; (iii) if the level of BMI-I mRNA or protein in the sample is indicative that the patient may benefit from enhanced prophylaxis for graft versus host disease prior to transplantation with an allogeneic stem cell transplant, providing said enhanced prophylaxis; and

(iv) transplanting the patient with an allogeneic stem cell transplant.

22. A method according to Claim 20 or 21 wherein a reduced level of BMI-I mRNA or protein is indicative that the patient may benefit from enhanced prophylaxis for graft versus host disease prior to transplantation with an allogeneic stem cell transplant.

23. A method according to Claim 20 or 21 wherein the prophylaxis for graft versus host disease comprises T cell depletion.

24. A method according to any one of Claims 17 to 23 wherein the sample is a CD34⁺ cell sample or a peripheral blood mononuclear cell sample.

25. A method according to any of Claims 17 to 24 wherein the sample is collected from the patient at the time of diagnosis of chronic myeloid leukemia.

26. A method according to any of Claims 17 to 25 wherein the level of BMI-I mRNA is measured.

27. A method according to Claim 26 wherein the level of BMI-I mRNA is measured by quantitative real time PCR.

28. A method according to Claim 18 wherein the alternative therapy comprises administration of a tyrosine kinase inhibitor, preferably imatinib mesylate.

29. Use of a reagent which selectively identifies BMI-I mRNA or protein in the assessment of the suitability to receive an allogeneic stem cell transplant of a patient having chronic myeloid leukemia, or the assessment of whether a patient selected to be transplanted with an allogeneic stem cell transplant for treatment of chronic myeloid leukemia should receive enhanced prophylaxis for graft versus host disease prior to being transplanted with the transplant.

30. Use according to Claim 29 wherein the reagent is one or more oligonucleotides.

31. Use according to Claim 30 in a quantitative real time PCR.

32. Use of a reagent which selectively identifies BMI-I mRNA or protein in the manufacture of a composition for aiding in assessing the suitability to receive an allogeneic stem cell transplant of a patient having chronic myeloid leukemia, or the assessment of whether a patient selected to be transplanted with an allogeneic stem cell transplant for treatment of chronic myeloid leukemia should receive enhanced prophylaxis for graft versus host disease prior to being transplanted with the transplant.

33. A method of improving the outcome of an allogeneic stem cell transplant in a patient, which patient is transplanted with the allogeneic stem cell transplant as a therapy for chronic myeloid leukemia, the method comprising administering an agent which is capable of increasing the expression or function of BMI-I mRNA or protein in the patient.

34. A method according to Claim 33 wherein the compound consists of or comprises a polynucleotide encoding BMI-I .

35. A method according to Claim 33 or 34 wherein the patient is administered the compound prior to or at substantially the same time as being transplanted with the allogeneic stem cell transplant.

36. A system comprising:

(i) a reagent for determining the level of BMI-I mRNA or protein in a sample from a patient and; either, (ii) a reagent for determining the level of BCR-ABL mRNA or protein in a sample from a patient; or

(iii) a reagent for determining the level of a further marker in a sample from a patient, which further marker is indicative of a particular outcome for a patient with chronic myeloid leukemia.

37. A system according to Claim 36 wherein the further marker is proteinase-3 mRNA or protein.

38. A method for identifying a compound useful in increasing the life expectancy of a patient in the chronic phase of chronic myeloid leukemia, the method comprising the steps of a) determining whether a test compound is capable of suppressing production of, or activity of, BMI-I in a sample from a patient with chronic myeloid leukemia and b) selecting a compound which is capable of suppressing production of, or activity of, BMI-I in a patient with chronic myeloid leukemia or in a sample from such a patient.

39. A method for identifying a compound useful in improving the outcome of an allogeneic stem cell transplant in a patient, which patient is transplanted with the allogeneic stem cell transplant as a therapy for chronic myeloid leukemia, the method comprising the steps of a) determining whether a test compound is capable of increasing production of, or activity of, BMI-I in a sample from a patient with chronic myeloid leukemia and b) selecting a compound which is capable of increasing production of, or activity of, BMI-I in a patient with chronic myeloid leukemia or in a sample from such a patient.