WO2008091679A2 - Method and kit for detection of mbcr/abl gene fusion and c-kit (cd117) - Google Patents

Abstract

Methods and kits for the identification of the major bcr/abl gene fusion and c-kit (CDl 17) expression in patients having or suspected of having a proliferative disorder characterized by the presence of the Mbcr/abl gene fusion and c-kit (CDl 17) expression are disclosed. The method simultaneously detects the presence of Mbcr/abl gene fusion and c-kit (CDl 17) in a biological sample using nucleic acid probes that hybridize to the BCR and ABLl genes, and detects c-kit(CDl 17) positivity. The method and kit can be used to predict the therapeutic efficacy of agents are inhibitors of tyrosine kinase, the BCR/ABL fusion protein c-kit (CDl 17).

Description

METHOD AND KTT FOR DETECTION OF

MBCR/ABL GENE FUSION AND C-KIT (CD117)

RELATED APPLICATIONS

This application claims priority under 35 U.S. C. § 119(e) to U.S. provisional Application Serial No.60/897,161 filed January 24, 2007, the entirety of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Chronic myeloid (or myelogenous) leukemia (CML) is characterized by new and genetically different marrow stem cells that produce increased numbers of myeloid cells (the white blood cells that normally engulf foreign matter and fight infection). The increase in CML tumor stem cells appears to suppress the normal blood cell precursors. Gale, R.P., 1991, "Chronic myelogenous leukemia: Molecule to man," Henry Ford Hospital MedicalJournal, 39:2:108-111. Although the disease involves a pluripotent stem cell, it is the myeloid lineage that usually predominates.

CML is dependent on a genetic alteration that results in a fusion between the ABLl gene on chromosome 9 and the BCR gene on chromosome 22. The genetic alteration is called the Philadelphia chromosome (Ph or Ph¹) for the city where it was first described. Nowell, PC and Hungerford, DA, 1960, "A minute chromosome in human chronic granulocytic leukemia," Science, 132:1497. At first this aberration was perceived as a deletion, but with the advent of better karyotypic resolution and chromosome staining methods, it was recognized as a reciprocal exchange of material between chromosomes 9 and 22, designated as t(9;22)(q34.1 ;ql 1.2) to identify the breakpoints. Rowley, J. D., 1973, "A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa staining," Nature, 243:290-293. The corresponding molecular alteration consists of a reciprocal translocation so that most or all of the oncogene ABLl moves from 9q34 to the BCR gene on chromosome 22 into a region known as the breakpoint cluster region (her). Bartram, C.R., et al., 1983, 'Translocation of c-abl oncogene correlates with the presence of a Philadelphia chromosome in chronic myelocytic leukemia," Nature, 306:277-280. The break mABLl is variable, although highly restricted within the gene, invariably 5' to the second exon, whereas the her breakpoint in CML may vary over the entire gene. The portion of the gene where the breaks are most common is within a 5.8 kb region known as major bcrβdbcr). Heisterkamp etal, 1988, 'The first BCR gene intron contains breakpoints in Philadelphia chromosome positive leukemia," Nucleic Acids Research, 16:21 : 10069-10081.

Although the Ph chromosome is found in more than 90 % of CML cases, related alterations mark some of the remaining cases of the disease. Variant or complex rearrangements that include other chromosomal sites as well as breaks at 9q34 and 22qll.2 are observed in approximately 4 to 8% of cases. Fitzgerald, 1991 , "Complex Ph Translocations in Chronic Myeloid Leukemia," Cancer Genet Cytogenet, 55: 129- 131 ; De Braekeleer, 1986, "Variant Ph translocations in CML: Is there an uneven geographic distribution?" Cancer Genet Cytogenet, 20:185-186. The remaining, Ph-negative cases comprise less than 5 % of all CML, since more rigorous criteria for discrimination from other myeloid dysplasias have been established, van der Plas et al, 1991 , "Review of clinical, cytogenetic, and molecular aspects of Ph-negative CML," Cancer Genet Cytogenet, 52:143-156.

However, as many as 50% of CML patients who test negative for the Ph chromosome by conventional cytogenetics have the Mbcr/abl rearrangement, indicating a molecular insertion of ABLl (a masked or cryptic translocation) into the BCR gene. Goldman, 1990, "Molecular biology and treatment of chronic myelogenous leukemia," Curr. Opin. Oncology, 2:49-54; Dobrovic et al, 1991, "Molecular Diagnosis of Philadelphia negative CML using the polymerase chain reaction and DNA analysis: clinical features and course of M-bcr negative and M-bcr positive CML," Leukemia, 5:3: 187-190; van der Plas et al., 1991 , "Review of clinical, cytogenetic, and molecular aspects of Ph-negative CML," Cancer Genet Cytogenet, 52:143-156. In these cases molecular means are required to identify the marker. Clinical and laboratory differences suggest that patients lacking the Mbcr/abl rearrangement at both levels may indeed have myeloproliferative disorders other than CML. First International Workshop on Chromosomes in Leukemia, 1977: "Chromosomes in Pin- positive chronic granulocytic leukaemia," Br. J. Haematol, 39:305-309, 1978.; Dobrovic et al., 1991, Leukemia, 5:3:187-190; Kurzrock and Talpaz, 1991, 'The molecular pathology of chronic myelogenous leukaemia," British Journal ofHaematology, 79: 1 :34-37. Detection of the Mbcr/abl rearrangement in Ph-negative CML patients or those with variant chromosomal translocations illustrates the greater resolving power of molecular-based techniques, the gene rearrangement can be detected with probe studies in situ or by Southern blot or PCR, but not by conventional cytogenetics. Kurzrock etal., \ 988, 'The molecular genetics of Philadelphia chromosome-positive leukemias," The New England J. of Medicine, 319:15 :990-998; Morris et al, 1990, "Ph-negative chronic myeloid leukemia: Molecular analysis of ABL insertion into M-BCR on chromosome 22," Blood, 76: 1812-1818.

The incidence of adult CML in Europe and the US is approximately 1 per 100,000, accounting for 20-30% of all leukemia cases. Carabasi, M.H., 1993, "Chronic myelogenous leukemia," Cancer Investigation, 11:4:408-419.

The Ph chromosome also is observed in other diseases, in a small proportion (3-10%) of children with Acute Lymphocytic Leukemia (ALL), and in approximately 20 to 25 % of adult ALL cases where it is strongly associated with poor survival. It is even less frequent (<2%) in Acute Myeloid Leukemia (AML) and these cases may represent examples of CML where the chronic phase was undetected or very brief Among the ALL cases where the Ph chromosome is present, the same molecular rearrangement found in CML has been reported in about 50% of children and near 50% of adults. Dreazen et al, 1988, "Molecular biology of chronic myelogenous leukemia," Seminars in Hematology, 25: 1 :35-49; Kantarjian et al, 1993, "Chronic myelogenous leukemia: A concise update," Blood, 82:3:691-703; Tanzer et al, 1991, "Leukemia myelbide chronique et syndromes myeloproliferatifs chronic myelogenous leukemia and myeloproliferative syndromes," J. of Experimental and Clinical Hermatology, 197-200. However, other investigators have found it in fewer than 25% of adult or childhood cases. Westbrook et al, 1992, "Clinical significance of the BCR-ABL fusion gene in adult acute lymphoblastic leukemia: A cancer and leukemia group B study (8762)," Blood, 80: 12:2983-2990.; Suryanarayan et al, 1991 , "Consistent involvement of the BCR gene by 9;22 breakpoints in pediatric acute leukemias," Blood, 77:324. In the remaining Ph-positive cases, although the chromosomal breakpoint appears identical, at the molecular level the break in the BCR gene is proximal on chromosome 22 (nearer to the centromere) and is designated as the minor breakpoint cluster region (mbcr).

CML, ALL and AML also may be characterized by markers in addition to Ph, including c-kit (CDl 17). Kindler et al , 'Εfficacy and safety of imatinib in adult patients with c-kit positive acute myeloid leukemia", 2004, Blood, 104(10): 3644-54; Malagola et al. , "Imatinib mesylate in the treatment of c-kit positive acute myeloid leukemia: is this the real target?' 2005, Blood, 105(2): 904-5. Cluster of differentiation (CD) molecules are markers on the cell surface, as recognized by specific sets of antibodies, used to identify the cell type, stage of differentiation and activity of a cell. C-kit (CDl 17) is the membrane receptor for stem cell factor (SCF), also known as "steel factor" or "c- kit ligand." Steel factor is a polypeptide that activates bone marrow precursors of a number of blood cells, but its receptor is also present on other cells. C-kit (CD-I 17) mutations in the interstitial cells of Cajal in the digestive tract are believed to be the key to gastrointestinal stromal tumors (GISTs).

SUMMARY OF THE INVENTION

The invention comprises methods, compositions and kits for the rapid and accurate identification of the major bcr/abl {Mbcr/abl) gene fusion and c-kit (CDl 17) amplification status in patients having or suspected of having a lymphoproliferative disorder characterized by the presence of the Mbcr/abl gene fusion and c-kit (CD 117) expression. The present method comprises simultaneously detecting both the presence of Mbcr/abl gene fusion and c-kit (CD 117) amplification in a single biological sample of patients having or suspected of having CML or another lymphoproliferative disorder using nucleic acid probes that hybridize to the BCR gene and the ABLl gene, together with a probe or antibody specific for c-kit (CD 117) expression.

The present method generally comprises the following steps: (a) disposing a biological sample obtained from a patient on a substrate using standard cytogenetic procedures; (b) if necessary, converting the DNA in the sample from double-stranded to single-stranded by denaturation; (c) contacting the sample with nucleic acid probes complementary to BCR and ABLl gene sequences, and optionally with nucleic acid probes complementary to a c-kit gene sequence, wherein the probes are labeled directly or indirectly with a detectable label; (d) incubating under conditions favorable to the hybridization of the probe DNA sequences and the genomic DNA sequences; and (e) determining the presence and relative positions of the detectable labels.

In a preferred embodiment, the method of the present invention comprises the following steps: (a) fixing a biological sample, preferably comprising whole cells such as peripheral blood cells or bone marrow, of a patient having or suspected of having CML or other lymphoproliferative disorder to a substrate, preferably a microscope slide; (b) converting the DNA in the sample from double-stranded to single-stranded by denaturation; (c) contacting the sample with DNA probes complementary to BCR and ABLl gene sequences, wherein the probes complementary to the BCR gene are tagged with one member of a first binding pair, and the probes complementary to the ABLl gene are tagged with one member of a second binding pair, and wherein the first and second binding pairs are different; (d) contacting the sample with DNA probes complementary to the c-kit (CDl 17) sequence, wherein the probes are either directly labeled or may be indirectly labeled, e.g., being tagged with one member of a third binding pair, wherein the third binding pair may be the same as the first or second binding pair, or may be a different binding pair; (e) incubating under conditions favorable to the annealing of the probe DNA sequences and the genomic DNA sequences; (f) optionally washing off the unannealed probe; (g) contacting the hybridized probes with fluorescently-tagged ligands comprising the other members of the first, second and, if applicable, third binding pairs, wherein the fluorescent label on the ligand belonging to the first binding pair is different from the label on the ligand belonging to the second binding pair (i.e., the labels fluoresce in different colors), and wherein the fluorescent label on the third binding pair may be the same as the label on the first or second binding pair, or may be a different label; (h) optionally staining the nonhybridized remainder of the DNA with a fluorescent counterstain in a third color; and (h) observing the slide, e.g., using epifluorescence illumination on a microscope or a bright field microscopy, to determine the relative positions of the fluorescent labels. The positions and colors of the fluorescent signals are indicative of the presence of the Philadelphia chromosome and of c-kit (CD 117) activation or expression.

The kit of the invention comprises (a) DNA probes comprising a DNA sequence complementary to the BCR gene tagged with a first detectable label or with one member of a first binding pair, a DNA sequence complementary to the ABLl gene tagged with a second different detectable label or with one member of a second different binding pair; and a DNA probe comprising a DNA sequence complimentary to the c-kit gene tagged directly with a detectable label or with a member of a third binding pair, (b) detection reagents for detection of hybridized probes; and (c) optionally, a counterstain for staining non-hybridized DNA. The kit optionally may contain reagents for denaturation and/or washing. In an alternative embodiment, the present method and kit may include an antibody specific for c-kit (CD 117) rather than a DNA probe. In a preferred embodiment, an antibody directed against the c-terminal domains of the c-kit (CD 117) protein is used, which recognizes both wild type and mutant forms of the c-kit (CD 117) protein.

In a preferred embodiment, the Mbcr DNA probes comprise overlapping cosmid clones which hybridize to the centromeric (5¹) side of the major breakpoint cluster region Mbcr within the BCR gene, and the abl DNA probes comprise four overlapping cosmid clones which hybridize to the telomeric (3') side of the ABLl gene. The c-kit probes preferably constitute a sequence that hybridizes to at least a portion of the c-kit (CD-I 17) gene.

In a currently preferred aspect, the present method and kit utilize in situ hybridization (ISH), preferably fluorescence in situ hybridization (FISH), to detect the presence of the Ph chromosome and c-kit (CD 117) expression

The present invention can be used for the diagnosis and/or monitoring of disease progression for lymphoproliferative disorders characterized by the presence of the Ph chromosome and c-kit (CD 117), including CML, AML and ALL, and for predicting the efficacy of certain therapeutic agents for treating such disorders. In one aspect, the present method and kit can be used to predict the efficacy of therapeutic agents which are inhibitors of a tyrosine kinase enzyme, the BCR/ABL fusion protein and c-kit (CDl 17), such as imatinib mesylate (Gleevec®), dasatinib (Sprycel™) or nilotinib (Tasigna™).

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 A shows the location ofhybridizatbn of the probes used in the present method to the BCR gene on chromosome 22 and the ABLl gene on chromosome 9; Figure 1 B shows the location ofhybridization of the probes used in the present method to the fused mbcr/abl site on chromosome 22.

Figures 2A-B show the specific location of the BCR and ABL genes on both 22ql 1.2

(BCR) and on 9q34 (ABL). Figure 2C shows the relationship of probes of the present invention in normal patient interphase nuclei. Figure 2D shows the relationship of probes of the present invention in a "fusion" configuration in tumor interphase nuclei. Figure 3 A shows the relationship of the c-kit gene probe on 4q 12 in relation to the centromere.

Figure 3B shows the relationship of the probes of the present invention in tumor interphase nuclei

DETAILED DESCRIPTION OF THE INVENTION

There is some discrepancy throughout the scientific literature regarding the precise nomenclature for certain genes and regions of genes. The majority of the human genome research community utilizes the gene symbols and map assignments in the Human Genome Database (GDB) (Johns Hopkins University), which are used herein as the standard. According to this standard, the ABLl gene is assigned to human chromosome 9q34.1

Heisterkamp et al., 1982, "Chromosomal localization of human cellular homologues of two viral oncogenes," Nature, 299:5885:747-749; Heisterkamp et al., 1988, 'The first BCR gene intron contains breakpoints in Philadelphia chromosome positive leukemia," Nucleic Acids Research, 16:21 :10069-10081; Hanish et al., 1991, "Application ofmethylase-limited partial Not! cleavage for a long-range restriction map of the human ABL locus," Genomics, 10:3:681-685 . The BCR gene is assigned to human chromosome 22qll. Groffen et al, 1984, 'Thiladelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22," Cell, 36:1:93-99; Cvoce et al, 1987, "Mapping of four distinct fiCft- related loci to chromosome region 22qll: order of BCR loci relative to chronic myelogenous leukemia and acute lymphoblastic leukemia breakpoints," Proc. Natl. Acad. Sd USA, 84:20:7174-7178. The regions within the BCR and ABLl genes containing the breakpoints for CML are herein designated Mbcr and abl, respectively. The fusion of these two regions as a result of a chromosome 9 and chromosome 22 translocation event in CML is herein designated Mbcr/abl.

Regardless of the form of treatment, remission and ultimately cure depend on elimination of the Ph-positive cells which show biologically different proliferative capacity. Goldman, 1990, "Molecular biology and treatment of chronic myelogenous leukemia," Curr. Opin. Oncology, 2:49-54; Kantarjian et al, 1993, "Chronic myelogenous leukemia: A concise update," Blood, 82:3:691-703. Monitoring for the absence ofor reduction in the frequency of the Ph chromosome is generally accepted as the appropriate method for patient follow-up. Treatment response can be monitored in terms of the presence or absence of the Ph chromosome.

Certain types of CML, as well as some other lymphoproliferative disorders are characterized by expression of c-kit (CD 117) proto-oncogene in addition to the Ph chromosome. C-kit (CD 117) is a tyrosine kinase receptor which is expressed in a wide variety of tumor cells. Therapeutic agents that inhibit c-kit (CD 117) as well as the bcr-able fusion protein are effective for treating these disorders. Such therapeutic agents include imatinib mesylate (Gleevec®), dasatinib (Sprycel™) and nilotinib. These agents may be more effective in those patients who are both Ph positive and c-kit positive. Moreover, the present test can identify patients that are Ph⁺ and c-kit negative, and Ph^" and c-kit positive, who also may benefit from therapy with these agents.

The present invention comprises methods, compositions and kits for the rapid and accurate identification and co-localization of the major bcr/abl (Mbcr/abl) gene fusion and c-kit (CDl 17) amplification status in a single biological sample obtained from patients having or suspected of having a lymphoproliferative disorder, including CML, AML or ALL. The present method comprises simultaneously detecting the presence of and co- localizing Mbcr/abl gene fusion and c-kit (CD 117) amplification in a single biological sample of a patients having or suspected of having CML or another proliferative disorder using nucleic acid probes that hybridize to the BCR gene and the ABLl gene, together with a probe or antibody specific for c-kit (CDl 17) expressioa The biological sample may be whole cells and preferably is cultured or uncultured bone marrow or peripheral blood cells.

In one aspect, the present method generally comprises the following steps: (a) disposing a biological sample obtained from a patient on a substrate using standard cytogenetic procedures; (b) if necessary, converting the DNA in the sample from double-stranded to single- stranded by denaturation; (c) contacting the sample with nucleic acid probes complementary to BCR end ABLl gene sequences and with a nucleic acid probe complementary to a c-kit gene sequence, wherein the probes are labeled with a detectable label; (d) incubating under conditions favorable to the hybridization of the probe DNA sequences and the genomic DNA sequences; and (e) determining the relative positions of the detectable labels. As used herein, the term "label" or "labeled" refers to any atom or moiety which can be used to provide a detectable (preferably, quantifiable) signal, and which can be attached to a polynucleotide, primer or probe. A wide variety of label and conjugation techniques, including direct and indirect labeling, are known and are reported extensively in both the scientific and patent literature. Examples of labels that can be used include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. The nucleic acid probes used in the present invention may be either directly labeled, e.g., using quantum dots or fluorescent compounds linked directly to the probe; or indirectly labeled, e.g., wherein a first member of a binding pair (such as streptavidin-biotin) is linked to the probe, and the label is attached to the second member of the binding pair. Preferred detectable labels for use in the present invention include quantum dots and fluorescent compounds. Direct and indirect labeling techniques and fluorophores useful as detectable labels are taught, for example, in U.S. Patent No. 7,316,931. The use of quantum dots for labeling biological compounds is taught, for example, in U.S. 7,235,361.

In a preferred embodiment, the method of the present invention comprises the following steps: (a) fixing a biological sample preferably comprising peripheral blood cells or bone marrow of a patient having or suspected of having CML to a substrate, preferably a microscope slide; (b) converting the DNA in the sample from double-stranded to single- stranded by denaturation; (c) contacting the sample with a DNA probe complementary to BCR and ABLl gene sequences, wherein the probe complementary to the BCR gene is tagged with one member of a first binding pair, and the probe complementary to the ABLl gene is tagged with one member of a second binding pair, and wherein the first and second binding pairs are different; (d) contacting the sample with a DNA probe complementary to a c-kit gene sequence, wherein the probe is either directly labeled or labeled with one member of a third binding pair; (e) incubating under conditions favorable to the annealing of the probe DNA sequences and the genomic DNA sequences in the patient sample; (f) optionally washing off the unannealed probe; (g) contacting the hybridized (annealed) probes with fluorescently-tagged ligands comprising the other members of the first and second (and if necessary, third) binding pairs, wherein the fluorescent label on the ligand belonging to the first binding pair is different from the label on the ligand belonging to the second binding pair (i.e., the labels fluoresce in different colors), the label on the third binding pair may be the same as the label on the first or second binding pair; (g) optionally staining the nonhybridized remainder of the DNA with a fluorescent counterstain in a third color; and (h) observing the slide, e.g., using epifluorescence illumination on a microscope or bright field microscopy, to determine the relative positions of the fluorescent labels. The colors of the fluorescent signals are indicative of the presence of the Philadelphia chromosome and of c-kit expressioa

Although any detection method can be used, including conventional cytogenetics, Southern Blot, or PCR-based methods, in situ hybridization (ISH) is preferred. ISH combines molecular and cytogenetic strengths. The ISH approach permits identification of chromosome aberrations in uncultured interphase nuclei, facilitating rapid detection in frequencies similar to those found after short term culture and chromosome harvest procedures. ISH can be applied to archival samples and to small numbers of cells. ISH techniques are well- known to those skilled in the art. See, e.g., Jin and Lloyd, "In situ hybridization: methods and applications," J ClinLabAnal, 1 l(l):2-9 (1997); Charuruks and Voravud, "In situ hybridization: a new tool in molecular medicine," J Med Assoc Thai, 79(6):374-81 (1996).

hi a preferred aspect, the present method and kit comprise the use of cosmid probe sets, one set containing a portion of the 5' end of the BCR gene; the other set containing sequences from the extreme 3' end of the ABLl gene, each linked with a different fluorophor, so that fusion or close approximation of two differently colored signals can be visualized. A separate probe is constructed for detecting c-kit (CD 117); that probe may be any sequence capable of hybridizing with all or a portion of the c-kit gene. The c-kit probe may be labeled directly, e.g. using quantum dot technology, or indirectly, e.g., using a fluorophore attached to one member of a binding pair. The present method is rapid, sensitive, and semiquantitative. It permits detection of the Mbcr/abl rearrangement and c-kit expression when a dividing cell population is unavailable, and has the added advantage of localizing the alteration to the tumor cell lineage.

The use of two-color FISH for only Mbcr/abl detection is known. Tkachuk et al, 1990, "Detection of bcr-abl fusion in chronic myelogenous leukemia by in situ hybridization," Science, 250:4980:559-562. Two color FISH has since been used to detect cryptic translocations and thus identifies between 2 and 3 % of CML cases that are not diagnosable by conventional metaphase analysis. Seong et al., 1993, "Detection of variant Ph-positive chronic myelogenous leukemia involving chromosomes 1 , 9 and 22 by fluorescence in situ hybridization," Cancer Genet Cytogenet, 65:100-103; Chen et al, 1993,

'Identification of masked and variant ph (complex type) translocations in CIVIL and classic ph in AML and ALL by fluorescence in situ hybridization with the use of bcr/abl cosmid probes," Cancer Genet Cytogenet, 70:103-107; Dewald etal., 1993, 'The application of fluorescent in situ hybridization to detect Mbcr/abl fusion in variant ph chromosomes in CML and ALL," Cancer Genet Cytogenet, 71:7-14. In metaphase preparations the same probes also can detect the mbcr of ALL; no fusion product is seen, but there is reversal of the expected signal on one set of the 9 and 22 chromosomes. Dewald et al, 1993, ibid. FISH is uniquely capable of displaying certain aberrations, such as the cases of cryptic translocation with the fusion gene at 9q34 rather than 22qll, and a duplication of Mbcr/abl resulting from mitotic recombination. Hagemeijer et al, 1993, 'Translocation of BCR to chromosome 9: A new cytogenetic variant detected by FISH in two Ph-negative, BCR-positive patients with chronic myeloid leukemia," Genes, Chromosomes & Cancer, 8:237-245.

FISH also has been used for determining c-kit mutations. Debiec-Rychter, et al, 2001, "Chromosomal aberrations in malignant gastrointestinal stromal tumors: correlation with c-KIT gene mutation," Cancer Genet Cytogenet; 128(1): 24-30 and Beghini, A., et al, 2000, 'Trisomy leading to duplication of a mutated KIT allele in acute myeloid leukemia with mast cell involvement," Cancer Genet Cytogenet; 119(1):26-31. However, the simultaneous detection and/or co-localization of the Mbcr/abl gene fusion and c-kit (CD 117) expression in a single patient sample is new.

In one aspect the kit of the invention comprises (a) DNA probes comprising at least one probe comprising a DNA sequence complementary to the BCR gene tagged with a first detectable label or with one member of a first binding pair, at least one probe comprising a DNA sequence complementary to the ABLl gene tagged with a second different detectable label with one member of a second different binding pair, and at least one probe comprising a DNA sequence complementary to the c-kit (CD 117) gene either directly labeled or tagged with one member of a third binding pair, (b) detection reagents for detection of hybridized probes; and (c) optionally, a counterstain for staining non-hybridized DNA The kit optionally may contain reagents for denaturation and/or washing

In a preferred embodiment, the Mbcr DNA probe comprises overlapping cosmid clones which hybridize to the centromeric (5*) side of the major breakpoint cluster region (Mbcr) within the BCR gene; and the abl DNA probe comprises four overlapping cosmid clones which hybridize to the telomeric (3') side of the ABLl gene. The c-kit DNA probe preferably comprises a sequence which hybridizes to all or a portion of the c-kit gene (4ql2). DNA probes useful in the present method and kit are those that hybridize to areas of the BCR and ABLI gene located on either side of the major breakpoint cluster regions: The following probe/primer sequences are preferred for use in the present invention:

MBCR Probes

Probes for BCLl Region

Probe Set 1 : A BAC clone proximal to the MTC region of the BCLl locus on 1 IqI 3 is generated by using the following primers 5 'AAGGTGTGAGGATCACTGGS' (SEQ ID NO. 1), 5ΑGCTCATGGGGGCTATT3' (SEQ ID NO. 2) derived from the Dl 1S1337 by screening a BAC library. In the nuclei carrying the translocation, this probe remains on the der(l 1) chromosome and generates a fusion or co-localizing signal in combination with the IGH probe from region 3.

Probe Set 2: A BAC clone telomeric to the BCLl gene region (is generated by using the following primers 5¹TTTCTGGGTGTGTCTGAATS' (SEQ ID NO. 3), 5'ACACAGTTGCTCT AAAGGGT3' (SEQ ID NO. 4) derived from the FGF3 gene. In the nuclei carrying the translocation, this probe segregates to the der(l 4) chromosome in translocations involving all the MTC, mTCl and mTc2 breakpoints within the BCLl locus on 1 IqI 3, and results in a fusion or co-localizing signal with the IGH probe from region 4.

Probes for IGH Region

Probe Set 3: A BAC clone covering the probe 3 region of the IGH gene is generated using the following primers derived from the sub-telomeric V region of the IGH gene to screen the BAC library: 5 TGTTTG AAG AAGGG AGTCGT3' (SEQ ID NO. 5); 5'CCCACTCCATGTCTTCTGTTS' (SEQ ID NO. 6). In the nuclei carrying the translocation, this probe segregates to the der(l 1) chromosome and generate a fusion or co- localizing signal in combination with probe 1.

Probe Set 4: A BAC clone covering the probe 4 region of the IGH gene is generated using the following primers derived from the C region to screen the library: 5'CCACTCAGTGTGACCTGGAGCGAAS' (SEQ ID NO. 7); 5'CTCCCCTGGCTTTTCTGGAACTGG3' (SEQ ID NO. 8). In the nuclei carrying the translocation, this probe remains on the der(14) chromosome and generates a fusion or co- localizing signal in combination with the relevant BCLl probe.

Probes for MYC Region

Probe Set 5: A BAC clone centromeric to all the three classes of breakpoints in the MYC region are generated using the following primers 5'GGCAAATTGGTTTAAGTGGAS' (SEQ ID NO. 9); 5'ACAGGGGAATGGTTAACTGG 3' (SEQ ID NO. 10) derived from WI- 1120. In nuclei carrying the translocation, this probe remains on the der(8) chromosome and generates a fusion or co-localizing signal in combination with the IGH probe from region 3

Probe Set 6: A BAC clone telomeric to all the breakpoints in the MYC gene is generated from the exon 2 region of MYC gene using the following primers 5^•ATGCCCCTCAACGTTAGCTTC3^• (SEQ ID NO. 11); 5¹CAGAGTCGCTGCTGGTGGTS' (SEQ ID NO. 12). In the nuclei carrying the translocation, this probe segregates to the der(14) chromosome in all three class types of breakpoints (i.e., breakpoints involving the first intron, the first exon, or further centromeric region breakpoints) and results in a fusion or co-localizing signal with IGH probe from region 4.

ABLl Probes

Probes for IGH Region

Probe Set 7: A BAC clone covering the probe 3 region of the IGH gene is generated using the following primers derived from the sub-telomeric V region of the IGH gene to screen the BAC library: 5TGTTTGAAGAAGGGAGTCGT3¹ (SEQ ID NO. 13); 5'CCCACTCCATGTCTTCTGrTS¹ (SEQ ID NO. 14). In the nuclei carrying the translocation, this probe segregates to the der(l 1) chromosome and generates a fusion or co- localizing signal in combination with probe 2.

Probe Set 8: A BAC clone covering the probe 4 region of the IGH gene is generated using the following primers derived from the C region to screen the library: 5¹CCACTCAGTGTGACCTGGAGCGAAS' (SEQ ID NO. 15); 5¹CTCCCCTGGCrTTTCTGGAACTGGS¹ (SEQ ID NO. 16). In the nuclei carrying the translocation, this probe remains on the der(14) chromosome and generates a fusion or co- localizing signal in combination with the probe 2.

Primers from the Region Centromeric to BCL2 Gene:

Probe Set 9: A BAC clone covering the probe 1 region of the BCL2 gene was generated using the following primers from the region centromeric to BCL2 gene to screen the library: 5'CTTGCCTCCTAAGCCAGTTG3' (SEQ ID NO. 17);

5TGGATTCATTTCTGATCCA3' (SEQ ID NO. 18). This probe always remained on the der(18) chromosome in nuclei with translocations involving all three breakpoints to generate fusion or co-localizing signal resulting from the segregation of probe 3 from 14q32 to 18q21.

Primers from the Region Telomeric to BCL2 Gene:

Probe Set 10: A BAC clone covering the probe 2 region of the BCL2 gene was generated using primers from the region telomeric to BCL2 gene: 5TGGCCAGCAGCAGTGTCAAATAG3' (SEQ ID NO. 19),

5^IGGTGATCGTTAGGACTCCCA3¹ (SEQ ID NO. 20). This probe segregates to the der(14) chromosome in translocations involving all three breakpoints within the BCL2 gene and results in a fusion or co-localizing signal with probe 4.

Probes for MBCR or ABLl

Primers from the Region Telomeric to IGH-J Gene:

Probe Set 11 : A BAC clone covering the probe 3 region of the IGH gene was generated using the following primers derived from the sub-telomeric region of chromosome 14q. 5TGTTTGAAGAAGGGAGTCGT3' (SEQ ID NO. 21); 5'CCCA CTCCATGTC TTCTGTT3' (SEQ ID NO. 22). In the nuclei carrying this translocation, the probe segregates to the der(l 8) chromosome and generates a fusion or co-localizing signal in combination with probe 1 in t(14;18)(q32;q21) cases.

Primers from the Region Centromeric to IGH-J Gene

Probe Set 12: A BAC clone covering the probe 4 region of the IGH gene was generated using the following primers derived from the C region: 5'CCCTGGACGCTTTTCAAATAS' (SEQ ID NO. 23); 5'GACTCAGGCAAGGACAAAGC3^I (SEQ ID NO. 24). In the nuclei carrying the translocation, this probe will remain on the der(14) chromosome and generate a fusion or co- localizing signal in combination with probe 2 from 18q21 region in t(14;18)(q32;q21) cases.

C-kit (CD 117) Probes

Probes for detecting c-kit (FLT3) Internal Tandem Duplication (ITD) Mutations

Probe Set 13: The juxtamembrane domain of the c-kit (FLT3) receptor cDNAs were amplified by RT-PCR, and one of the primers was FAM labeled. Primer sequences were 5'GCAATTTAGGTATGAAAGCCAGC3^I (SEQ ID NO. 25) and

5'CTTTCAGCATTTTGACGGCAACCS' (SEQ ID NO. 26). The wild-type receptor led to a PCR product of 240 bp that was detected by Genescan analysis (ABI3700). c-kit (FLT3) ITD mutations were identified by the increased size of the PCR product. Heterozygous mutations were identified by the presence of both PCR products.

In the present method, five probe sets (a total often probes) preferably are used: any two MBCR probe sets (probe sets 1 -6) may be combined with any two ABLl probe sets (probe sets 7-10) or anytwo MBCR or ABLl probe sets (probe sets 11-12). The fifthprobe set is the C-kit probe set (probe set 13).

In a currently preferred aspect, the present method and kit utilize in situ hybridization (ISH), preferably fluorescence in situ hybridization (FISH), to detect the presence of the Philadelphia chromosome and c-kit (CD 117) expression. In an alternative embodiment, c-kit (CD 117) expression can be detected using a labeled polyclonal or monoclonal antibody specific for c-kit (CD 117) protein. Such antibodies have been disclosed. See, e.g., Langner etal, Eur. J Surg. Oncol, 30(8):847-50 (2004); Reed et al., CHn. Colorectal Cancer, 2(2): 119-22 (2002).

The present method and kit can be used for the diagnosis and/or monitoring of disease progression for lymphoproliferative disorders and for predicting and/or monitoring the efficacy of certain therapeutic agents for treating disorders characterized by the presence of the Ph chromosome and c-kit, including CML, AML and ALL. In one aspect, the present method and kit can be used to predict the efficacy of therapeutic agents which are inhibitors of tyrosine kinase, the BCR/ ABL fusion protein and c-kit (CDl 17), such as imatinib mesylate (Gleevec®), dasatinib (Sprycel™) and/or nilotinib. In a preferred aspect, the method and kit of the present invention utilize a set of DNA probes and detection reagents that yield an easily readable fluorescent signal, preferably a red fluorescent signal at the site of the BCR gene, a green fluorescent signal at the site of the ABLl gene, and a green fluorescent signal at the site of the c-kit gene, on a blue fluorescent background of nuclear or chromosomal DNA. Preferred probes are those having the sequences shown as SEQ ID NOs. 1-26. Most preferably, four probes specific for MBCR selected from probe sets 1-6 (SEQ ID NOs. 1-12) are combined with four probes specific for ABLl or for both ABLl or MBCR selected from probe sets 7- 12 (SE ID Nos. 13-24). For c-kit, probe set 13 (SEQ ID NOs. 25-26) is used. The method and kit yield these results on both metaphase chromosomes and interphase nuclei of bone marrow and peripheral blood cells. The c-kit label may be any color, however.

In a preferred embodiment, the method of the present invention comprises the following steps: (a) a biological sample preferably comprising whole cells, preferably peripheral blood cells or bone marrow of a patient having or suspected of having CML is fixed to a substrate, preferably a microscope slide, using standard cytogenetic procedures; (b) the DNA in the sample is converted from double-stranded to single-stranded by denaturation at about 70 °C using a mixture of the 2OX SSC and Formamide Reagents supplied in the kit; (c) the labeled DNA probes complementary to BCR, ABLl and c-kit gene sequences are applied to the slide, which then is incubated under conditions favorable to the annealing of the probe DNA sequences and the genomic DNA sequences; (d) unannealed probe is washed off using a mixture of the 2OX SSC and Formamide Reagents, and the hybridized probes are detected by an indirect methodology using fluorescently-tagged ligands which bind to the labels on the DNA probes is and the remainder of the DNA is then stained with a fluorescent counterstain; and (e) the slide is observed by epifluorescence illumination on a microscope or bright field microscopy.

In a currently preferred embodiment, a kit according to the invention comprises (a) labeled DNA probes comprising DNA sequences complementary to the BCR gene on chromosome 22, the ABLl gene on chromosome 9 and the c-kit (CD 117) gene on chromosome 4 (location 4q 12); (b) detection reagents for fluorescent detection of hybridized probes; and (c) fluorescent counterstain. The kit optionally also may contain reagents for denaturation and/or washing. In a preferred embodiment, iheMbcr DNA probes comprise the sequences shown herein as SEQ ID NOs. 1-12, and the abl DNA probes comprise the sequences shown herein as SEQ ID NOs. 13-24, and the c-kit DNA probes comprise the sequences shown herein as SEQ ID NOs. 25 and 26.

The detection reagent permits simultaneous detection of hybridized Mbcr, abl and c-kit (CD 1 17) DNA sequences. In a preferred embodiment, the detection reagent comprises rhodamine-conjugated anti-digoxigenin antibody and fluorescein isotinocyanate- conjugated avidin. The digoxigenin-labeled Mbcr probe is detected by the anti-digoxigenin antibody, the anti-digoxigenin antibody binds with high affinity to the digoxigenin label on the probe, thereby immobilizing the fluorescent rhodamine at the site of the BCR gene. The biotin-labeled abl probe is detected by the avidin; the avidin binds with high affinity to the biotin label on the probe, thereby immobilizing the fluorescein at the site of the ABLl gene. Subsequent washing with phosphate buffered detergent (PBD) reagent in the kit removes unbound detection reagent. The c-kit probe can be labeled directly (e.g. using Q-dots), or indirectly (e.g., using the rhodamine-conjugated anti-digoxigenin antibody/digoxigenin binding pair or the fluorescein isotinocyanate-conjugated avidin/biotin binding pair).

The counterstain reagent utilized in a preferred embodiment of the present kit comprises DAPI (4',6'-diamidino-2-phenylindole) and, optionally, an antifade agent. DAPI stains the nuclear or chromosomal DNA uniformly by intercalating between bases in the DNA double helix. The antifade component retards photobleaching of the fluorescent signal.

Excitement of rhodamine, fluorescein, and DAPI by light from a mercury arc lamp in a fluorescence microscope results in the emission of red, green and blue light, respectively. The observer selects for these three colors by using the appropriate microscope filter, and simultaneously scores nuclei or metaphase chromosomes for the presence of red, green, or fused red-green signals on a blue background.

The method and kit of the present invention provide information about the number of major bcr/abl fusion events that are present in an assayed cell, and can be used in the diagnosis of patients who are suspected of having CML, AML, ALL or clinically similar lymphoproliferative disorders. The present method and kit can detect the appearance of a second Mbcr/abl fusion event, which often is characteristic of disease progression to theblastic phase of CML; therefore, they can be used as a clinical tool to determine whether a patient has entered the blastic phase, or to guide changes in therapy intended to address the blastic phase of the disease. They also may be used as a clinical tool use in the diagnosis, prognosis and/or treatment of other lymphoproliferative disorders, including acute lymphocytic leukemia (ALL) or acute myebid leukemia (AML). The DNA probe in the present method and kit detect the major her breakpoint, which is the site of translocation in CML and in AML or ALL cases that exhibit a Philadelphia chromosome. The Mbcr/abl fusion in such cases can be detected by the present assay.

The method and kit of the present invention also can be used to test individuals with clinical indications of lymphoproliferative disease in order to rule out CML.

The present invention is illustrated further by the following non-limiting Example.

EXAMPLE

Three hundred and forty-seven (347) samples of bone marrow from patients suspected of having CML were prepared and analyzed as described below. For each specimen, the Mbcr/abl fusion signals were recorded in 200 individual non-overlapping nuclei.

Three sets of slides were prepared, each set containing the test samples and three control samples: five (5) samples of each from normal, CML c-kit positive and CML c-kit negative patients. One set of the three specimens was denatured, hybridized and post- washed at the recommended temperatures, another set was assayed at -1°C from the recommended temperatures, and the third set was assayed at -2°C from the recommended temperatures. The sample were masked and then scored using the scoring criteria provided below. Each of the 9 slides (3 per patient sample) was evaluated for the presence of the Mbcr/abl fusion product in each of 200 interphase nuclei. When present, up to 20 metaphase spreads per slide were also evaluated for the fusion product.

Nuclei from the samples were fixed by standard cytogenetic procedures and applied to a microscope slide. The DNA was converted from double-stranded to single-stranded by denaturation at 70 °C using a mixture of the 2OX SSC and Formamide Reagents supplied in the kit. A mixture of labeled DNA probes complementary to the BCR and ABLl gene sequences prepared as described above were applied to the slide. The Mbcr DNA probes comprised three digoxigenin-labeled overlapping cosmid clones which hybridize to the centromeric (5') side of the major breakpoint cluster region (Mbcr) within the BCR gene (Probe Sets 1 and 2; SEQ ID NOs. 1-4). The abl DNA probes comprised four biotin- labeled overlapping cosmid clones which hybridize to the telomeric (3') side of the ABLl gene (Probe Sets 7 and 8; SEQ ID NOs. 13-16). C-kit probes comprising sequences complementary to a portion of the c-kit gene (Probe Set 13; SEQ ID NOs. 25 or 26), were labeled with biotin and applied to the slide.

The slides then were incubated under conditions favorable to the annealing of the probe DNA sequences and the genomic DNA sequences. Unannealed probe was washed off using a mixture of 2OX SSC, and Formamide Reagents. Detection reagents comprising fluorescently-tagged ligands which bind to the labels on the DNA probes were then applied to the slides to detect hybridized probes. The detection reagent contained both rhodamine-conjugated anti-digoxigenin antibody and fluoresceinisothiocyanate-conjugated avidin. The anti-digoxigenin antibody binds with high affinity to the digoxigenin label on the probe, thereby immobilizing the fluorescent rhodamine at the site of the BCR gene. The avidin binds with high affinity to the biotin label on the ABL and c-kit probes, thereby immobilizing the fluorescein at the site of the ABLl and c-kit genes. Subsequent washing with the PBD reagent in the kit removed unbound detection reagent.

The remainder of the DNA was then stained with a fluorescent counterstain, DAPI (4',6'-diamidino-2-phenylindole) and antifade was applied. The slide then was observed by epifluorescence illumination on a microscope. Excitement of rhodamine, fluorescein, and DAPI by light from a mercury arc lamp in a fluorescence microscope resulted in the emission of red, green and blue light, respectively. The DNA probes and detection reagents utilized yielded a red fluorescent signal at the site of the BCR gene and a green fluorescent signal at the site of the ABLl gene on both metaphase chromosomes and interphase nuclei of bone marrow and peripheral blood cells, on a blue fluorescent background of nuclear or chromosomal DNA.

The observer selected for these three colors by using the appropriate microscope filter, and simultaneously scored nuclei or metaphase chromosomes for the presence of red, green, or fused red-green signals on a blue background. The following criteria were used to interpret the results:

1. All nuclei which appear intact and likely to contain all chromosomal material were scored.

2. Only nuclei which are non-overlapping were scored. Nuclei that are touching but not overlapping may be scored if the point of contiguity does not contain signal.

3. A total of 200 randomly selected interphase nuclei were scored: 100 in each of two different areas of the slide.

4. The Mbcr signal appears red, while the abl and c-kit (CDl 17) signals appear green. The following signals should be scored as Mbcr/abl fusions:

4.1 A single spot of yellow, yellow-white, or white coloration.

4.2 An elongated spot which is red on one end, green on the other, and yellow, yellow-white or white in the center.

4.3 A red spot touching a green spot.

The presence of c-kit (CDl 17) expression is indicated by the presence of two green spots. Accordingly, the presence of both Mbcr/abl fusions and c-kit (CDl 17) expression is indicated by a yellow or white spot and two green spots. If neither is present, the slide will show two red and two green spots with no yellow or white spots.

The following criteria were used in scoring:

*A normal cell in G2 will show two sets of closely paired green dots and two sets of closely paired red dots. A fusion positive cell in G2 will show one set of closely paired green dots, one set of closely paired red dots and one set of closely paired fusion signals.

The percentage of apparent Mbcr/abl fusion positive cells expected in normal individuals is a mean of about 4.8% based on scoring 200 nuclei each in 144 Mbcr/abl fusion negative samples. This study indicated that >96% of Mbcr/abl fusion negative specimens will test between 0 and 11% (95% confidence).

The mean percentage of Mbcr/abl fusion positive cells found in CML-affected individuals was 84.9% based on scoring 200 nuclei each in 203 CML patient peripheral blood or bone marrow samples. This study also indicated that >92% of the Mbcr/abl fusion positive specimens will test between the cut-off value of >11% and 100% (95% confidence).

The analytical sensitivity of the method was determined from the standard deviation (S. D.) of 25 known normal blood specimens tested. This population had a mean of 3.8% fusion positive interphase nuclei with a S. D. of 1.5. Therefore, the analytical sensitivity of the present method and kit was determined to be about 2x1.5%, or 3.0% interphase nuclei showing Mbcr/abl fusions in a total population of at least 200 interphase nuclei scored.

Scoring methods which can be used in the present method include those disclosed by Signoretti et al, "Her-2-neu Expression and Progression Toward Androgen Independence in Human Prostate Cancer," J. Natl. Cancer Instil, 92(23): 1918-25 (2000); Gu et al, "Prostate stem cell antigen (PSCA) expression increases with high gleason score, advanced stage and bone metastasis in prostate cancer," Oncogene, 19: 1288-96 (2000).

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Claims

What is claimed is:

1. A method for simultaneously determining the presence of a translocation between a BCR and an ABLl gene and expression of a c-kit gene in a biological sample containing chromosomal material of a patient comprising the steps of:

a. converting the chromosomal material to single stranded DNA;

b. contacting the sample with at least one first nucleic acid probe which is complementary to the BCR gene and at lease one second nucleic acid probe which is complementary to the ABLl gene, wherein the first probe is tagged with a first detectable label and the second probe is tagged with a second detectable label, and wherein the first and second detectable labels are different;

c. contacting the sample with a third nucleic acid probe which is complementary to at least a portion of the c-kit gene, wherein the third probe is tagged with a third detectable label;

d. incubating under conditions sufficient to induce the probes to hybridize to the genes; and

e. determining the presence of the translocation and c-kit by observing the presence and location of the first, second and third detectable labels.

2. The method of claim 1 where in the biological sample is bone marrow or peripheral blood cells.

3. The method of claim 1 wherein the detectable labels are fluorescent labels.

4. The method of claim 1 wherein the probe complementary to the BCR gene is selected from the group consisting of SEQ ID NOs. 1-12.

5. The method of claim 1 wherein the probe complementary to the ABLI gene is selected from the group consisting of SEQ ID NOs. 13-24.

6. The method of claim 1 wherein the probe complementary to the c-kit gene is selected from the group consisting of SEQ ID NOs. 25 and 26.

7. The method of claim 1 wherein an indirect labeling technique is used,

wherein in step (b) the first probe is tagged with one member of a first binding couple and the second probe is tagged with one member of a second binding couple, wherein the first and second binding couples are different;

in step (c), the third probe is tagged with one member of a third binding couple; and

which, after step (d), comprises the following additional step:

contacting the sample with a first ligand comprising the other member of the first binding couple and a first detectable label, a second ligand comprising the other member of the second binding couple and a second detectable label, and a third ligand comprising the other member of the third binding couple and a third detectable label, wherein the first and second detectable labels are different.

8. The method of claim 7 wherein the first binding couple is digoxigenin- antidigoxigenin antibody, and the second binding couple is avidin- biotin.

9. A method for predicting the efficacy of a therapeutic agent which inhibits bcr-abl tyrosine kinase and c-kit in a patient who is positive for the Philadelphia chromosome abnormality comprising simultaneously determining the presence of the Philadelphia chromosome abnormality and c-kit expression in the chromosomal material of the patient according to the method of claim 1.

10. A kit for determining the presence of a translocation between a BCR and an ABLl gene and expression of a c-kit gene in a biological sample containing chromosomal material of a patient comprising:

a. at least one first nucleic acid probe which is complementary to the BCR gene and at least one second nucleic acid probe which is complementary to the ABLl gene, wherein the first probe is tagged with a first detectable label and the second probe is tagged with a second detectable label, wherein the first and second labels are different; and

b. at least one third nucleic acid probe complementary to the c- kit(CDl 17) gene, wherein the third probe is tagged with a third detectable label.

1 1. The kit of claim 10 wherein the first nucleic acid probe is selected from the group consisting of SEQ ID NOs. 1-12.

12. The kit of claim 10 wherein the second nucleic acid probe is selected from the group consisting of SEQ ID NOs. 13-24.

13. The kit of claim 10 wherein the third nucleic acid probe is selected from the group consisting of SEQ ID NOs. 25 and 26.

14. The kit of claim 10 wherein an indirect labeling technique is used, and wherein

the first probe is tagged with one member of a first binding couple and the second probe is tagged with one member of a second binding couple, wherein the first and second binding couples are different;

the third probe is tagged with one member of a third binding couple;

and which includes the following additional components: a first ligand comprising the other member of the first binding couple and a first detectable label, a second ligand comprising the other member of the second binding couple and a second detectable label, and a third ligand comprising the other member of the third binding couple and a third detectable label.