WO2008051817A1

WO2008051817A1 - Compositions and method for monitoring bcr-abl expression

Info

Publication number: WO2008051817A1
Application number: PCT/US2007/081870
Authority: WO
Inventors: Vija R. Modur; Charlene So; Yaping Shou; Alan K. Hatfield; Elisabeth Wehrle
Original assignee: Novartis Ag
Priority date: 2006-10-20
Filing date: 2007-10-19
Publication date: 2008-05-02

Abstract

The invention provides the parameters required to make high quality standards for use in monitoring bcr-abl expression in subjects and determine whether a patient with Ph+CML has achieved Major Molecular Response (MMR) that is associated with a low risk of disease relapse. The invention provides a way to generate a definition of this standard in actual terms, i.e. in bcr-abl transcript copy number. The standard for bcr-abl transcript copy number is objectively determined and measurable.

Description

COMPOSITIONS AND METHOD FOR MONITORING bcr-abl EXPRESSION

FIELD OF THE INVENTION

[01] This invention relates generally to the analytical testing of tissue samples in vitro, and more particularly to aspects of bcr-abl gene expression.

BACKGROUND OF THE INVENTION

[02] Chronic myeloid leukaemia (CML) is a clonal disease of the haematopoietic stem cell in which a reciprocal translocation, t(9;22)(q34;ql 1), forms the Philadelphia chromosome (Ph) and creates a novel fusion gene, bcr-abl. The bcr-abl gene expresses an activated tyrosine kinase that is central to the pathogenesis of CML. Imatinib mesylate (Gleevec®, Glivec®) is a tyrosine kinase inhibitor that blocks the kinase activity of bcr-abl, thus inhibiting the proliferation of Ph-positive progenitors.

[03] The frequency of major medical responses in patients with chronic myeloid leukaemia (CML) undergoing imatinib treatment is correlated with the gene expression of bcr-abl. Hughes TP et al, N. Engl. J. Med. 349(15): 1423-1432 (October 9, 2003). Hughes et al. measured levels of bcr-abl transcripts by a quantitative real-time polymerase chain reaction (PCR) assay. Results were expressed relative to the median level of the ratio of bcr- abl to bcr transcripts in the blood of 30 patients with untreated CML in chronic phase. In patients who had a complete cytogenetic remission, levels of bcr-abl/bcr transcript ratio after twelve months of treatment had fallen by at least 1000-fold (3 log) in many of those in the imatinib group. For patients who had a complete cytogenetic remission and a reduction in transcript levels of at least 3 log at 12 months, the probability of remaining progression-free was 100% at 24 months. The proportion of patients with CML who had a reduction in bcr- abl transcript levels of at least 3 log by 12 months of therapy was far greater with imatinib treatment than with an alternative treatment. Moreover, patients in the imatinib group with this degree of molecular response had a negligible risk of disease progression during the subsequent twelve months. Thus the response was durable.

[04] However, this standard (reduction of 3 log) is not well defined in terms of copy numbers. Attempts have been made to estimate transcript numbers using a real time PCR- based competitive technique so that transcript numbers can be expressed per μg of leukocyte RNA or as a ratio of bcr-abl/abl on a log scale, but not directly. See Hughes T et al, Blood 108(l):28-37 (July 1, 2006, Epub March 7, 2006). Use of ratios to express molecular response to Gleevac has several problems. Investigation of abl as the control gene has revealed substantial differences in stability of abl and bcr-abl between individual samples were observed upon storage. Moreover, at high transcript levels, i.e. in patients with CML at diagnosis or CML still predominantly Ph positive, the fact that the abl control also measures bcr-abl gives a spuriously high result and may therefore underestimate the bcr-abllabl ratio. Accordingly, there is a risk that CML patients who receive imatinib and respond initially lose their response because they have Ph-positive sub-clones characterized by the presence of one or other of a range of mutations in the bcr-abl kinase domain (KD) that codes for a specific amino-acid substitution. See, e.g., Growney JD et al, Blood 106(2):721-4 (July 15, 2005; Epub March 24, 2005). Although each assay may be consistent within any particular lab, the lack of complete consensus on assay design, reference standards, and reporting of results is a problem acknowledged by the CML community In response, Hughes T et al, Blood 108(l):28-37 (July 1, 2006, Epub March 7, 2006) recommend (1) using a conversionfactor whereby individual laboratories can express bcr-abl transcript levels on an agreed scale, (2) using serial real time quantitative PCR (RQ-PCR) results rather than bone marrow cytogenetics or fluorescence in situ hybridization (FISH) for the bcr-abl gene to monitor individual patients, and (3) detecting and reporting Ph-positive sub-populations bearing bcr- abl kinase domain mutations.

[05] Molecular response is an emerging therapeutic goal in CML therapy.

Accordingly, there continues to be a need for directly measuring bcr-abl transcripts to monitor response to treatment by tyrosine kinase inhibitors such as Gleevec®, to conduct surveillance for detecting subsequent increases in bcr-abl gene expression that can result in remission and to ensure that the patients receive the optimal dose of tyrosine kinase inhibitor treatment.

SUMMARY OF THE INVENTION

[06] The invention provides high quality reference standards for use in monitoring bcr- abl expression in subjects. The invention provides a definition of this standard in actual terms, i.e. in bcr-abl transcript copy number or in bcr-abl transcript level relative to an internal control (thus providing an equivalent of a bcr-abl transcript copy number based upon the transcript copy number of a control transcript). In several embodiments the internal control may be bcr transcript copy numbers or abl transcript copy number.

[07] The standard for bcr-abl transcript copy number is objectively determined and measurable. In one embodiment, the standard is based upon the standard provided by this invention..

[08] The standard pegs the copy number of bcr-abl transcript in patients at the time of achievement of MMR as 100 or fewer copies per microgram RNA from whole blood.

Accordingly, the standard advantageously provides an absolute number which can be used to calibrate different bcr-abl PCR tests from different laboratories, the number being about 18 copies per microgram RNA from patient blood. In one embodiment, the number is 17.91 ±

81.24 copies, or from 0 to 99.15 copies (i.e., less than 100). In another embodiment, the

17.91 ± 40.62 copies, or from 0 to 58.53copies (i.e., 59).

[09] Moreover, the new standard in the tests can be applied to monitor different patients without first applying a conversion factor for each specific test used for bcr-abl transcript monitoring.

[10] The invention thus usefully provides methods for monitoring the decrease of mRNA transcripts during treatment with an anti-CML therapy, such as administration of a bcr-abl modulating agent. In several embodiments, the bcr-abl modulating agent can be imatinib mesylate (Gleevec®/Glivec®) or nilotinib (Tasigna®). In one embodiment, the subject is tested every 3 months, to monitor achievement and maintenance of major molecular response (MMR). If the subject doesn't reach MMR, then the subject can be switched to an alternative therapy.

[11] In one aspect, the invention provides a way to express results in terms of major molecular response (MMR) that can be identified as the equivalent determined by the method of the invention of a >3 log reduction in bcr-abl transcript copy levels from a standardized baseline.

BRIEF DESCRIPTION OF THE DRAWINGS

[12] The drawing figures depict preferred embodiments by way of example, not by way of limitations. In the figures, like reference numerals refer to the same or similar elements. [13] FIG. 1 is a pictograph showing bcr-abl transcript numbers expressed as a log reduction of transcripts for patients responding to Gleevec®/Glivec® treatment. The left scale is a measure of the log reduction in bcr-abl transcript from baseline in the patients. The right scale is a measure of the number of leukaemia cells in a patient.

[14] FIG. 2 is a scatter plot of bcr-abl copy number in 181 patients on the IRIS trial at the time of achievement of MMR prior to 12 months on Glivec therapy.

[15] FIG. 3 is a pictograph showing the IRIS study design and patient status.

[16] FIG. 4 is a graph showing the survival without accelerated phase/blast crisis

(AP/BC) by molecular response at 12 months on first-line imatinib treatment.

[17] FIG. 5 is a graph showing the survival without AP/BC by molecular response at 18 months on first-line imatinib treatment.

[18] FIG. 6 is bar graph showing response without AP/BC at 60 months by molecular response at 12 and 18 months.

DETAILED DESCRIPTION OF THE INVENTION

[19] Rationale for the bcr-abl transcript assay aspect of the invention. The IRIS Phase

3 trial (see, EXAMPLE; Hughes TP et al, N. Engl. J. Med. 349(15): 1423-1432 (October 9, 2003); O'Brien SG et al, N. Engl. J. Med. 345:994-1004 (2003)) showed that (a) the molecular response indicates reduction in bcr-abl transcript levels; (b) the major molecular response (MMR) is >3-log reduction from a standardized baseline; (c) administration of Gleevec®/Glivec® yields high rates of MMR; and (d) MMR correlates with 5-year progression free and overall survival (see also, Druker BJF et al, J. Clin. Oncol 24(18S): 338s. Abstract 6506 (2006)).

[20] There is a correlation between bcr-abl transcript levels and leukaemia cells. See

FIG. 1. bcr-abl transcripts are both a cause of leukemia and a measure of disease and response to therapy. Branford S et al, Br. J. Haematol. 107(3): 587-99 (1999). When the level of bcr-abl transcripts is a low number, a major molecular response is achieved. Thus, a reduction of bcr-abl transcript levels correlates with a good outcome for anti-CML therapy. [21] It is the absolute bcr-abl transcript levels corresponding to the 3 -log reduction, not the relative reduction of bcr-abl, that is important. Thus, this bcr-abl transcript copy number allows the creation of a standard that can provide prognostic information in terms of major molecular response. Baccarani, et al, Blood (2006). See also, evidence from the IRIS study that it is the absolute number of bcr-abl transcripts and not the relative reduction in the number that is important. Hughes TP et al, N. Engl. J. Med. 349(15): 1423-1432 (October 9, 2003); O'Brien SG et al, N. Engl. J. Med. 345:994-1004 (2003). A rise in bcr-abl transcript levels 2-fold can signal resistance. Branford S et al, Blood 104(9): 2926-32 (2004). However, a rise may not be significant. Wang L et al, Haematologica 91(2): 235-9 (2006). [22] Accordingly, the aims of the PCR testing aspect of the invention are: (a) to optimize therapy with Gleevec®/Glivec®; (b) to monitor response with most sensitive technology available; (c) to detect minimal residual disease; (d) to obtain early indication of relapse (see, Asnafi V et al, Leukemia 20(5): 793-9 (2006)); and (e) to screen for resistance (Branford S et al, Blood 104(9): 2926-32 (2004)).

[23] The MMR rates improve with time on GleevecΦ/Glivec® therapy. Goldman J et al, Blood 106 (2005). MMR at 12 or 18 months correlates with 5-year progression-free survival and overall survival. Druker BJF et al, J. Clin. Oncol. 24(18S): 338s. Abstract 6506 (2006). No patient with CML in chronic phase who achieved an MMR at 12 or 18 months of Gleevec®/Glivec® therapy progressed to advanced disease after 5 years in the IRIS trial. See FIG. 5.

[24] In addition, bcr-abl transcript levels at time of complete cytogenetic response

(CCyR) predict duration of CCyR. Press RD et al, Blood 107(11): 4250-6 (2006). Thus, the level of bcr-abl mRNA at 12 months of Gleevec®/Glivec® therapy is a significant predictive laboratory marker of subsequent progression-free survival.

[25] It has been demonstrated that patients with CML and undetectable bcr-abl with therapy have lower rates of relapse compared with patients with detectable bcr-abl. Colombat M et al, Haematologica 91(2): 162-8 (2006).

[26] The European LeukemiaNet Consensus has issued a set of recommendations for response assessment in CML. Baccarani, et al, Blood (2006). The recommended schedule for response assessment is that: (a) beginning at diagnosis of chronic-phase CML, there should be a haematologic response evaluation every 2 weeks until complete haematologic response; (b) bone marrow cytogenetics should be performed every 6 months until CCyR, then every 12 months (see, Ross DM et al, Leukemia 20(4): 664-70 (2006)); (c) bcr-abl transcript level should be evaluated to confirm disease and establish baseline level; (d) in situ hybridization should be performed in cases of suspected Philadelphia chromosome-negative, Z?cr-αZ)/-positive disease or variant translocations; and (e) bcr-abl transcript levels should be measured at 3 -month intervals during therapy and continued even if results are negative (i.e., when bcr-abl transcripts are not detected). [27] However, until now the number of transcripts associated with a major molecular response was unknown. Here, we show that the mean bcr-abl copy number at the time of achievement of MMR in 181 patients on the IRIS trial is 95% of patients who achieve major molecular response for the first time prior to the first year in the IRIS trial have a value of bcr- abl transcript below 100 (99.1 rounded off to 100) copy numbers in 1 microgram of RNA from blood.

[28] Patient sample. As used herein, a "sample" means a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid {e.g., blood, plasma or serum) or the isolated nucleic acid or polypeptide derived therefrom. A sample from a human patient generally contains about 2 μg RNA/mL blood.

[29] Reference standards. In one embodiment, the standards used for assaying bcr-abl gene expression levels are produced using the Armored RNA® technology. WalkerPeach CR et al, Clin. Chem. 45(12):2079-85 (December 1999); Pasloske BL et al, J. Clin. Microbiol. 36(12):3590-4 (December 1998). Armored RNA® technology is a means for producing RNA that is protected from plasma ribonucleases, such that it is stable and resistant to degradation in plasma. Nevertheless, it can be used as a template for PCR assays. Accordingly, ribonuclease-resistant polynucleotides are used as RNA controls and calibrators. Moreover, the standard can be easily reformatted to allow evaluation of newly emerging assay formats. WalkerPeach CR et al, Clin. Chem. 45(12):2079-85 (December 1999). By use of this technology, the previously observed anomalies in differential bcr-abllabl transcript stability are advantageously obviated.

[30] Other polynucleotides that are resistant to ribonuclease are also known to those of skill in the art. See, PCR Primer, 2nd edition, eds. Dieffenbach & Dveksler. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003); Molecular Diagnostic PCR Handbook, by Louis H. NeI, John R. Crowther, Gerrit J. ViIj oen (Springer 2005) and PCR Technology: Current Innovations by Thomas Weissensteiner, Hugh G. Griffin, Annette M. Griffin (CRC Press 2004). Such polynucleotides can be used as reference standards in the measurement of bcr-abl transcripts by PCR assay.

[31 ] Decrease from a standardized baseline defined by the number of bcr-abl transcripts associated with chronic myelogenous leukaemia (CML). In one embodiment, the standard baseline is operationally defined by the serial or other dilution of a cell line known to have a high level of bcr-abl transcript (such as the K562 cell line, Klein et al, Int. J. Cancer 18(4):421-31 (October 15, 1976)) in white blood cells of normal subjects (i.e., subjects without CML).

[32] Three control genes that appear to be suitable for bcr-abl quantitation are bcr, abl and β-glucuronidase (gusB). Hughes T et al, Blood 108(l):28-37 (July 1, 2006, Epub March

7, 2006).

[33] abl has been used by many investigators in studies evaluating minimal residual disease (MRD) in patients treated with imatinib. Cortes J et al., Clin. Cancer Res. 11 : 3425-

3432 (2005). Further investigation of abl as the control gene has revealed comparable mean stability of abl and bcr-abl, but substantial differences between individual samples were observed upon storage, van der Velden VH et al, Leukemia 8: 884-886 (2004). At high transcript levels, i.e., in patients with CML at diagnosis or CML still predominantly

Philadelphia chromosome-positive, the fact that the abl control also measures bcr-abl gives a spuriously high result and may therefore underestimate the bcr-abl/abl ratio.

[34] bcr was initially investigated as a control gene since it has a similar expression level and stability to that of bcr-abl. Subsequent experiments did indeed confirm that bcr degrades at the same rate as bcr-abl. Hughes TP & Branford S, Monitoring Disease

Response, in: MeIo JV, Goldman JM, eds. Myeloproliferative disorders (Springer- Verlag;

2006). bcr was the control gene selected for the IRIS study. Hughes TP et al, N. Engl. J.

Med. 349(15): 1423-1432 (October 9, 2003).

[35] Wang and colleagues (Wang YL et al, J. Molecular Diagnostics (2006) have argued in favor of gus B, which in contrast to bcr and abl has the theoretical advantage that it is not rearranged in the leukemia cell population. Thus though abl is currently the most widely used control gene, bcr and gusB are equally suitable.

[36] Clinical decision making based on bcr-abl transcript assay results. In one embodiment, the resulting measurement is a characterization of molecular response during therapy. Branford S & Hughes T, Methods MoI Med. 125: 69-92 (2006); Hughes T et al,

Blood 108(l):28-37 (July 1, 2006, Epub March 7, 2006). The measurement can be (a) bcr-abl transcript levels continuing to decline; (b) undetectable; (c) stable or (d) rising. In another embodiment, bcr-abl transcript levels are measured at 3-month intervals.

[37] It is to be appreciated that certain aspects, modes, embodiments, variation and features of the invention are described below in various levels of detail in order to provide a substantial understanding of the present invention. In general, such disclosure provides useful methods for the monitoring of subjects in need thereof. Accordingly, the various aspects of the present invention relate to diagnostic and theranostic methods and kits to identify individuals predisposed to disease or to classify individuals with regard to drug responsiveness, side effects, or optimal drug dose. The methods and kits are useful for studying the aetiology of diseases, studying the efficacy of drug targeting, predicting individual susceptibility to diseases, and predicting individual responsiveness to drugs targeting the gene product. Accordingly, various particular embodiments that illustrate these aspects follow.

[38] Definitions. The definitions of certain terms as used in this specification are provided below. Definitions of other terms may be found in the glossary provided by the U.S. Department of Energy, Office of Science, Human Genome Project

(http://www.ornl.gov/sci/techresources/Human Genome/glossary/). In practicing the present invention, many conventional techniques in molecular biology, microbiology and recombinant DNA are used. These techniques are well-known and are explained in, e.g., Current Protocols in Molecular Biology, VoIs. I-III, Ausubel, ed. (1997); Sambrook et ah, Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989); DNA Cloning: A Practical Approach, VoIs. I and II, Glover D, ed. (1985); Oligonucleotide Synthesis, Gait, ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, eds. (1985); Transcription and Translation, Hames & Higgins, eds. (1984); Animal Cell Culture, Freshney, ed. (1986); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; the series, Methods in Enzymol. (Academic Press, Inc., 1984); Gene Transfer Vectors for Mammalian Cells, Miller & Calos, eds. (Cold Spring Harbor Laboratory, New York, 1987); and Methods in Enzymology, VoIs. 154 and 155, Wu & Grossman, and Wu, eds., respectively. [39] The term "about" refers to the inherent limitations in the equipment used to measure chemical quantities and the differences in how those of skill in the art make those measurements. A number of polynucleotides is "about" a certain number {e.g., 18) when a measurement of the number of polynucleotides in a sample would be considered to be acceptably close to the number by one of skill in the art. See, PCR Primer, 2nd edition, eds. Dieffenbach & Dveksler. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003); Molecular Diagnostic PCR Handbook, by Louis H. NeI, John R. Crowther, Gerrit J. Viljoen (Springer 2005) and PCR Technology: Current Innovations by Thomas Weissensteiner, Hugh G. Griffin, Annette M. Griffin (CRC Press 2004). Such polynucleotides can be used as reference standards in the measurement of bcr-abl transcripts by PCR assay.

[40] The term "biological sample" is intended to include, but is not limited to, e.g. , tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

[41] The term "clinical response" means any or all of the following: a quantitative measure of the response, no response, and adverse response (i.e., side effects). For a description of Sokol groups regarding staging and prognosis in chronic myelogenous leukaemia, see Sokal JE et al., Semin. Hematol. 25: 49-61 (1988). See also, Hasford J et al, J Natl Cancer Inst 90:850-8 (1998).

[42] The term "clinical trial" means any research study designed to collect clinical data on responses to a particular treatment, and includes, but is not limited to phase I, phase II and phase III clinical trials. Standard methods are used to define the patient population and to enrol subjects.

[43] The term "effective amount" of a compound is a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, for example, an amount which results in the prevention of, or a decrease in the symptoms associated with, a disease that is being treated. The amount of compound administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. Typically, an effective amount of the compounds of the present invention, sufficient for achieving a therapeutic or prophylactic effect range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. A preferred dosage ranges from about 0.0001 mg per kilogram body weight per day to about 1,000 mg per kilogram body weight per day. Another preferred dosage ranges from about 0.01 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. The compounds of the present invention can also be administered in combination with each other, or with one or more additional therapeutic compounds.

[44] The U.S. Food & Drug Administration recommended dosage of Gleevec®

(imatinib mesylate) is 400 mg/day for adult patients in chronic phase CML and 600 mg/day for adult patients in accelerated phase or blast crisis. The recommended Gleevec dosage is 260 mg/m²/day for children with Ph⁺ chronic phase CML recurrent after stem cell transplant or who are resistant to interferon-alpha therapy. The recommended dosage of Gleevec is 400 mg/day or 600 mg/day for adult patients with unresectable and/or metastatic, malignant GIST. [45] In the EXAMPLE and in the IRIS study, patients with newly diagnosed CML received 400 mg of imatinib per day. Hughes TP et al, N. Engl. J. Med. 349(15): 1423-1432 (October 9, 2003); O'Brien SG et al, N Engl. J. Med. 348:994-1004 (2003). [46] As used herein, "expression" includes but is not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and mRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function. [47] The term "bcr-abl modulating agent" is any compound that alters (e.g., increases or decreases) the expression level or biological activity level of the expression oϊ bcr-abl as compared to the expression level or biological activity level in the absence of the bcr-abl modulating agent.

[48] In one embodiment, the bcr-abl modulating agent is Gleevec®. Imatinib mesylate

(Gleevec®, Glivec®) is a tyrosine kinase inhibitor that blocks the kinase activity of BCR- ABL, thus inhibiting the proliferation of Ph-positive progenitors. Buchdunger E et al, Cancer Res. 56:100-4 (1996). Druker BJ et al, Nat. Med. 2:561-6 (1996). See, U.S. Pat. No. 5,521,184 and European Patent EP 0 564 409. See also, Druker B et al, Nature Medicine, 2(5) (May 1996).

[49] In another embodiment, the "bcr-abl modulating agent" is nilotinib (Tasigna®)

Weisberg E et al, Cancer Cell 7:129-141 (2005). Nilotinib is an oral tyrosine kinase inhibitor that targets BCR-ABL, KIT, and platelet derived growth factor receptor (PDGFR). Other bcr-abl modulating agents are available. La Rosee P, Cancer Res. 62:7149-7153 (2002); Shah NP et al, Science 305:399-401 (2004); Drucker BJ, N. Engl. J. Med. 354:2594- 2596 (June 15, 2006). See, published PCT international patent application WO 2004/005281 and U.S. Pat. No. 7,169,791. See also, published PCT international patent applications WO 2007/015870 and WO 2007/015871.

[50] In another embodiment, the "bcr-abl modulating agent" is dasatinib (BMS-

354825; Sprycel®). [51] In addition, other tyrosine kinase inhibitors may have an effect upon the methods of the invention. For example Growney et al. suggest that PKC412 may be a useful therapeutic agent for c-KIT positive malignancies harbouring the imatinib-resistant activation mutations. Growney JD et al, Blood 106(2):721-4 (July 15, 2005; Epub March 24, 2005). PKC412 is a staurosporine derived tyrosine kinase inhibitor that targets PKC, KDR, VEGF- R2, PDGFRα, FLT3 and c-KIT. Fabbro D et al, Anticancer Drug Des. 15:17-28 (2000). PKC412 has been shown to be effective against the fusion protein FIPlLl -PDGFRα with mutations in the kinase domain that are resistant to imatinib. Cools J et al, Cancer Cell 3:459-469 (2003). Since inhibitors of D816V/Y c-kit mutations would potentially have therapeutic activity in systemic mast cell disease (SMCD) and acute myeloid leukaemia (AML), Growney et al evaluated the effectiveness of PKC412 against a panel of c-kit mutations identified in SMCD, AML and gastrointestinal stromal tumour (GIST) patients. Growney JD et al, Blood 106(2):721-4 (July 15, 2005; Epub March 24, 2005). Note that the tyrosine kinase inhibitor Gleevec® is efficacious in the majority of GIST patients harbouring c-kit mutations

[52] The term "medical condition" includes, but is not limited to, any condition or disease manifested as one or more physical and/or psychological symptoms for which treatment is desirable, and includes previously and newly identified diseases and other disorders. In one embodiment, the medical condition is chronic myelogenous leukaemia (CML). In another embodiment, the medical condition is acute myelogenous leukaemia (AML).

[53] The term "population" may be any group of at least two individuals. A population may include, e.g., but is not limited to, a reference population, a population group, a family population, a clinical population, and a same sex population. In one embodiment, the population is the set of subjects with a Philadelphia (Ph) chromosome. [54] The term "reference standard population" means a population characterized by one or more biological characteristics, e.g., drug responsiveness, genotype, haplotype, phenotype, etc. Hughes et al. used a reference standard population of 30 patients with untreated CML in chronic phase to determine a median level oibcr-abl transcripts. Hughes TP et al, N. Engl J. Med. 349(15): 1423-1432 (October 9, 2003).

[55] The term "reference standard gene expression profile" is the pattern of expression of one or more gene observed in either a reference standard population or a single subject prior to administration of a compound. In one embodiment, the profile is the expression of bcr-abl/bcr as provided by Hughes TP et al, N. Engl. J. Med. 349(15): 1423-1432 (October 9, 2003).

[56] The term "subject" means that preferably the subject is a mammal, such as a human, but can also be an animal, including but not limited to, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like) and laboratory animals (e.g., monkeys such as cynmologous monkeys, rats, mice, guinea pigs and the like). [57] The administration of an agent or drug to a subject or patient includes self- administration and the administration by another. It is also to be appreciated that the various modes of treatment or prevention of medical conditions as described are intended to mean "substantial", which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved. [58] The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[59] Detection of Gene Expression. An exemplary method for detecting the presence or absence of nucleic acid of the invention (e.g., bcr-abl, abl or bcr) in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound capable of detecting an expressed polynucleotide (e.g., mRNA) that encodes a mutant polypeptide of the invention, such that the presence of mutant gene is detected in the biological sample. A compound for detecting an expressed polynucleotide can be a labelled nucleic acid probe capable of hybridizing to an expressed polynucleotide. The nucleic acid probe can be, for example, a full-length mutant nucleic acid or a portion thereof, such as an oligonucleotide of at least 5,15, 30, 50,100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to the an expressed polynucleotide. Other suitable probes for use in the diagnostic assays of the invention are described herein.

[60] For example, in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of mutant genomic DNA include Southern hybridizations. See, for example Molecular Cloning A Laboratory Manual, Second Ed., Sambrook, Fritsch & Maniatis, ed. (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II, Glover DN ed. (1985); Oligonucleotide Synthesis, Gait MJ ed. (1984); Nucleic Acid Hybridization, Hames BD & Higgins SJ, eds., 1984). Techniques for the detection of gene expression of the genes described by this invention include, but are not limited to Northern blots, RT-PCT, real time PCR, primer extension, RNase protection, RNA expression profiling and related techniques. In one embodiment, the technique for detecting gene expression includes the use of a gene chip. The construction and use of gene chips are well known in the art. See, U.S. Pat Nos. 5,202,231; 5,445,934; 5,525,464; 5,695,940; 5,744,305; 5,795,716 and 5,800,992. See also, Johnston M, Curr. Biol, 8:R171-174 (1998); Iyer VR et al, Science, 283:83-87 (1999) and Elias P, "New human genome 'chip' is a revolution in the offing" Los Angeles Daily News (October 3, 2003).

[61] In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound capable of detecting the expressed polynucleotide of the invention, such that the presence of polynucleotide is detected in the biological sample, and comparing the presence of polynucleotide in the control sample with the presence of polynucleotide in the test sample. [62] In one embodiment, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. Guidance for sample collection and preparation of blood samples is provided by Hughes T et al, Blood 108(l):28-37 (July 1, 2006, Epub March 7, 2006).

[63] Amplifying a Target Gene Region. The target regions may be amplified using any oligonucleotide-directed amplification method, including but not limited to polymerase chain reaction (PCR). (U.S. Pat. No. 4,965,188), ligase chain reaction (LCR) (Barany et al, Proc. Natl. Acad. Sci. USA, 88:189-193 (1991); published PCT patent application WO 90/01069), and oligonucleotide ligation assay (OLA) (Landegren et al, Science, 241 :1077-1080 (1988)). Qligonucleotides useful as primers or probes in such methods should specifically hybridize to a region of the nucleic acid that contains or is adjacent to the polymorphic site. Typically, the oligonucleotides are between 10 and 35 nucleotides in length and preferably, between 15 and 30 nucleotides in length. Most preferably, the oligonucleotides are 20 to 25 nucleotides long. The exact length of the oligonucleotide will depend on many factors that are routinely considered and practiced by the skilled artisan. In one embodiment, the target comprises a mutation, e.g., polymorphism of the invention, in particular, bcr-abl. [64] Procedure for bcr-abl transcript assay. PCR is a method that amplifies short

DNA sequences (100 to 600 bases) within a longer double-stranded DNA molecule. To detect messenger RNA (mRNA transcripts), the method was extended using reverse transcriptase to convert mRNA into complementary DNA (cDNA). For use with bcr-abl transcripts, see Mensink E et al, Br. J. Haematol. 102(3): 768-74 (1998); Preudhomme C et al, Leukemia 13(6): 957-64 (1999); van der Velden VH et al, Leukemia 17(6): 1013-34 (2003). [65] Real-time PCR is a quantitative approach in which the polymerase reaction is monitored in the early stages while there is a linear relationship between amplified DNA and the detected signal used to measure it. RQ-PCR testing for bcr-abl is performed using laboratory-developed assays. Current assays can detect one bcr-abl-positi\e cell in a million normal cells.

[66] Other nucleic acid amplification procedures. Other known nucleic acid amplification procedures may be used to amplify the target region including transcription- based amplification systems. (U.S, Pat. No. 5,130,238; EP 0 329 822; U.S. Pat. No. 5,169,766, published PCT patent application WO 89/06700) and isothermal methods (Walker et al, Proc. Natl. Acad. ScI, USA, 89:392-396 (1992).

[67] An alternative method for determining the level of mRNA corresponding to a marker of the present invention in a sample involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental embodiment set forth by Mullis, U.S. Pat. No. 4,683,232); ligase chain reaction, Barany (1991), supra; self-sustained sequence replication, Guatelli et al, Proc, Natl. Acad. Sci, USA, 87:1874-1878 (1990); transcriptional amplification system, Kwoh et al, Proc. Natl. Acad. Sci. USA, 86:1173-1177 (1989); Q-Beta Replicase, Lizardi et al, Biol. Technolog , 6: 1197 (1988); rolling circle replication, U.S. Pat. No. 5,854,033; or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well-known to those of skill in the art. These detection schemes are especially useful for the detection of the nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10-30 nucleotides in length and flank a region from about 50-200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

[68] As noted above, RT-PCR (real-time quantitative PCR) is one way to assess gene expression levels, e.g., of genes of the invention (e.g., those containing SNPs and polymorphisms of interest). The RT-PCR assay utilizes an RNA reverse transcriptase to catalyze the synthesis of a DNA strand from an RNA strand, including an mRNA strand. The resultant DNA may be specifically detected and quantified and this process may be used to determine the levels of specific species of mRNA. One method for doing this is known under the Trademark TAQMAN (PE Applied Biosystems, Foster City, CA) and exploits the 5' nuclease activity of AMPLITAQ GOLD™ DNA polymerase to cleave a specific form of probe during a PCR reaction. This is referred to as a TAQMAN™ probe. See Luthra et al., Am. J. Pathol., 153: 63-68 (1998)). The probe consists of an oligonucleotide (usually ~20 mer) with a 5 '-reporter dye and a 3' -quencher dye. The fluorescent reporter dye, such as FAM (6-carboxyfluorescein), is covalently linked to the 5' end of the oligonucleotide. The reporter is quenched by TAMRA (6-carboxy-N,N,N',N'-tetramethylrhodamine) attached via a linker arm that is located at the 3' end. See Kuimelis et al, Nucl. Acids Symp. Ser., 37: 255- 256 (1997) and Mullah et al., Nucl. Acids Res., 26(4): 1026- 1031 (1998)). During the reaction, cleavage of the probe separates the reporter dye and the quencher dye, resulting in increased fluorescence of the reporter.

[69] The accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. See Heid et al., Genome Res., 6(6): 986-994 (1996). Reactions are characterized by the point in time during cycling when amplification of a PCR product is first detected rather than the amount of PCR product accumulated after a fixed number of cycles. The higher the starting copy number of nucleic acid target, the sooner a signifϊcant increase in fluorescence is observed, (Gibson et al, Genome Res., 6: 995-1001 (1996)).

[70] When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by Fδrster-type energy transfer. See Lakowicz et al, J. Biol. Chem., 258:4794-4801 (1983)).

[71] The gene expression of a biomarker gene can be quantified in a test sample using

TAQMAN™ Method Quantitating Gene Expression Level. Briefly, TAQMAN™ (PE Applied Biosystems, Foster City, CA) exploits the 5' nuclease activity of AMPLITAQ GOLD™ DNA polymerase to cleave a specific form of probe during a PCR reaction, i.e., TAQMAN™ probe. (Luthra et al, Am. J. Pathol, 153:63-68 (1998)). The probe consists of an oligonucleotide (usually «20 mer) with a 5 '-reporter dye and a 3' quencher dye. The fluorescent reporter dye, such as FAM (6-carboxyfluorescein), is covalently linked to the 5' end of the oligonucleotide. The reporter is quenched by TAMRA. (6-carboxy-N,N,N',N'- tetramethylrhodamine) attached via a linker arm that is located at the 3' end. (Kuimdis et al, Nucl. Acids. Symp. Ser., 37:255-256 (1997); Mullah et al, Nucl Acids Res., 26(4): 1026- 1031 (1998)). During the reaction, cleavage of the probe separates the reporter dye and the quencher dye, resulting in increased fluorescence of the reporter. The accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. (Heid et al, Genome Res., 6(6):986-994 (1996)). Reactions are characterized by the point in time during cycling when amplification of a PCR product is first detected rather than the amount of PCR product accumulated after a fixed number of cycles. The higher the starting copy number of nucleic acid biomarker, the sooner a significant increase in fluorescence is observed. (Gibson et al, Genome Res., 6:995-1001 (1996)). When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by Fδrster-type energy transfer. (Lakowicz et al, J. Biol Chem., 258:4794-4801 (1983)). During PCR, if the biomarker of interest is present, the probe specifically anneals between the forward and reverse primer sites. The 5 '-3' nucleolytic activity of the AMPHTAQ GOLD™ DNA polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the biomarker. The probe fragments are then displaced from the biomarker, and polymerization of the strand continues. This process occurs in every cycle and does not interfere with the exponential accumulation of product. The 3' end of the probe is blocked to prevent extension of the probe during PCR. The passive reference is a dye included in the TAQMAN™ buffer and does not participate in the 5' nuclease assay. The passive reference provides an internal reference to which the reporter dye signal can be normalized during data analysis. Normalization is necessary to correct for fluorescent fluctuations due to changes in concentration or volume. Normalization is accomplished by dividing the emission intensity of the reporter dye by the emission intensity of the passive reference to obtain a ratio defined as the R_n (normalized reporter) for a given reaction tube. The threshold cycle or C_t value is the cycle at which a statistically significant increase in ΔR_n is first detected. On a graph of R_n vs. cycle number, the threshold cycle occurs when the sequence detection application begins to detect the increase in signal associated with an exponential growth of PCR product. To perform quantitative measurements serial dilutions of a cRNA (standard) are included in each experiment in order to construct a standard curve necessary for the accurate and fast mRNA quantification. In order to estimate the reproducibility of the technique the amplification of the same cRNA sample may be performed multiple times.

[72] Other Methods for Detecting Gene Transcription. Other technologies for measuring the transcriptional state of a cell produce pools of restriction fragments of limited complexity for electrophoretic analysis, such as methods combining double restriction enzyme digestion with phasing primers (see, e.g., EP 0 534858 Al), or methods selecting restriction fragments with sites closest to a defined mRNA end. See, e.g., Prashar et al, Proc. Natl. Acad. ScL, USA, 93(2):659-663 (1996)).

[73] Other methods statistically sample cDNA pools, such as by sequencing sufficient bases, e.g., 20-50 bases, in each of multiple cDNAs to identify each cDNA, or by sequencing short tags, e.g., 9-10 bases, which are generated at known positions relative to a defined mRNA end pathway pattern. See, e.g., Velculescu, Science, 270:484-487 (1995). The cDNA levels in the samples are quantified and the mean, average and standard deviation of each cDNA is determined using by standard statistical means well-known to those of skill in the art. Bailey NTJ, Statistical Methods In Biology, Third Edition (Cambridge University Press, 1995).

[74] For in situ methods, mRNA does not need to be isolated from the cells prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the marker. As an alternative to making determinations based on the absolute expression level of the marker, determinations may be based on the normalized expression level of the marker. Expression levels are normalized by correcting the absolute expression level of a marker by comparing its expression to the expression of a gene that is not a marker, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes, such as the actin gene or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., a. patient sample, to another sample or between samples from different sources.

[75] Alternatively, the expression level can be provided as a relative expression level.

To determine a relative expression level of a marker gene, the level of expression of the marker is determined for 10 or more samples of normal versus disease biological samples, preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the marker. The expression level of the marker determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that marker. This provides a relative expression level. The choice of the cell source is dependent on the use of the relative expression level. Using expression found in normal tissues as a mean expression score aids in validating whether the marker assayed is specific (versus normal cells). In addition, as more data is accumulated, the mean expression value can be revised, providing improved relative expression values based on accumulated data.

[76] Kits of the Invention. The kit can comprise, e.g., (1) an oligonucleotide, e.g., a detectably-labelled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention; or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention. [77] The kit can also comprise, e.g. , a buffering agent, a preservative or a protein- stabilizing agent. The kit can further comprise components necessary for detecting the detectable-label, e.g., an enzyme or a substrate. The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. In a preferred embodiment, such kit may further comprise a DNA sample collecting means. The kits of the invention may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit. In several embodiments, the use of the reagents can be according to the methods of the invention. In one embodiment, the reagent is a gene chip for determining the gene expression of relevant genes.

[78] The following EXAMPLE is presented in order to more fully illustrate the preferred embodiments of the invention. This EXAMPLE should in no way be construed as limiting the scope of the invention, as defined by the appended claims.

EXAMPLE

LONG-TERM BENEFIT OF IMATINIB FOR PATIENTS NEWLY DIAGNOSED WITH

CHRONIC MYELOGENOUS LEUKEMIA (CML) IN CHRONIC PHASE

[79] Study design. IRIS was a prospective, multicenter, international, open-label, phase

3, randomized trial. Patients with newly diagnosed, chronic-phase chronic myeloid leukaemia (CML) were randomized to each treatment arm; N = 553 in each.

[80] Patients in the imatinib group received 400 mg once daily. Patients in the interferon alpha (IFN-α) + cytarabine (Ara-C) group received gradually escalating doses of

IFN-α (target dose: 5 million units per meter squared of body surface per day). Once the maximum dose of IFN-α was achieved, subcutaneous low-dose Ara-C was added at a dose of

20 mg per meter squared per day (maximal daily dose: 40 mg) for 10 days every month.

Patients were allowed to cross over to the other arm if they had no response, had a loss of response, had an increase in the white-cell count, or could not tolerate treatment. See FIG. 3.

[81] The primary study end point was event-free survival.

[82] 382 (69%) of the 553 patients randomized to the imatinib arm remained on imatinib after a median follow-up of 60 months. Sixteen (3%) of the 553 patients randomized to IFN-α plus Ara-C remain on IFN-a plus Ara-C after a median follow-up of 60 months.

[83] Discontinuation of first-line imatinib. TABLE 1 shows the discontinuations during the course of treatment. TABLE 1

Imatinib n (%)

Discontinued first-line treatment 157 (28)

Side effects / CML-unrelated deaths 32 (6)

Lack of efficacy / progression 60 (11)

Other reason 65 (12)

Cross-over to IFN and discontinued 14 (3)

[84] In those 181 patients who achieved MMR prior to 12 months of therapy with

Glivec in the IRIS trial, the bcr-abl transcript number was below 100 (99.1 rounded off to 100) copies per microgram of blood RNA. The mean bcr-abl copies/mL was 17.91 and the standard deviation was 40.62. Furthermore, the median copy numbers was 3. See, FIG. 2.

TABLE 2 bcr-abl Copy Number

# of Mean Standard Maximum 75th Median 25th Minimum atients Deviation percentile percentile

181 17.91 40.62 272 15 3 1 0

[85] FIG. 4 is a graph showing the survival without AP/BC by molecular response at 12 months on first-line imatinib treatment. Molecular response was evaluated every 3 months after a CCyR was obtained using real-time quantitative polymerase chain reaction (RQ-PCR). The ratio of bcr-abl/bcr transcripts was measured and results were expressed as "log reductions" below a standardized baseline derived from a median bcr-abl/bcr value obtained from 30 untreated patients with chronic-phase CML. Major molecular response (MMR) was defined as >3 log reduction of bcr-abl/bcr transcripts. . For 95% of individuals, this MMR is associated with a bcr-abl copy number below 100. No patient with CCyR and MMR by 12 months progressed to AP/BC.

[86] FIG. 6 is bar graph showing response without AP/BC at 60 months by molecular response at 12 and 18 months. EQUIVALENTS

[87] The present invention is hot to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the invention. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the invention, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present invention is to be limited only by the terms of the appended claims along with the full scope of equivalents to which such claims are entitled.

Claims

CLAIMS We claim:

1. A reference standard for the measurement of bcr-abl transcripts by PCR assay, comprising an set of isolated bcr-abl polynucleotides, wherein the number of polynucleotides in the set are 17.91 ± 81.24 polynucleotides.

2. The standards of claim 1, wherein the number of polynucleotides in the set are 17.91 ± 40.62 polynucleotides.

3. The standards of claim 1, wherein the number of polynucleotides in the set are about 18 polynucleotides

4. The standards of claim 1, wherein the polynucleotides are selected from the group consisting of RNA or a ribonuclease-resistant polynucleotide.

5. A method for monitoring chronic myelogenous leukaemia (CML) in a subject who receiving an anti-CML treatment, comprising the steps of:

(a) determining the absolute copy number of bcr-abl transcript in a blood, plasma or serum sample taken from the subject, and

(b) monitoring the course of anti-CML treatment, wherein a decrease of the absolute copy number of bcr-abl transcript or maintenance of the absolute copy number of bcr-abl transcript at a level of below 100 copies per microgram RNA from whole blood indicates that the anti-CML therapy is successful.

6. The method of claim 5, wherein the anti-CML treatment is the administration of a composition of matter selected from the group consisting of imatinib or a salt thereof or nilotinib or a salt thereof.

7. The method of claim 5, wherein the anti-CML treatment is imatinib mesylate or nilotinib.

8. The method of claim 5, wherein the determination of the level of bcr-abl transcript in the subject is performed by reverse transcriptase-polymerase chain reaction (RQ-PCR) amplification and quantitation of transcript levels.

9. The method of claim 5, wherein the determination of the level of bcr-abl transcript in the subject is performed by a polymerase chain reaction (PCR) with the reference standard of claim 1.

10. The method of claim 5, wherein the monitoring of the patients on imatinib or nilotinib therapy further comprises the step of setting the optimal dose of imatinib or nilotinib therapy.

11. The method of claim 5, wherein the monitoring of the patients on imatinib or nilotinib therapy further comprises the step of assessing response to novel therapies against Philadelphia chromosome (Ph⁺)-chronic myelogenous leukaemia (CML).