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Methods of analyzing chromosomal translocations using fluorescence in situ hybridization (fish)Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidMethods of analyzing chromosomal translocations using fluorescence in situ hybridization (fish) description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070166749, Methods of analyzing chromosomal translocations using fluorescence in situ hybridization (fish). Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application is a divisional of U.S. patent application Ser. No. 10/146,658, filed on May 14, 2002, which claims priority to U.S. Provisional Application Nos. 60/291,121, filed May 14, 2001; 60/337,653, filed Nov. 8, 2001; and 60/357,195, filed Feb. 13, 2002. Each of these applications is incorporated herein by reference in its entirety for all purposes. BACKGROUND [0003] Chromosomal rearrangements can take a variety of forms including exchanges of portions of one chromosome with another chromosome (translocations), deletion of sections of chromosomes, insertions of new material into the chromosome, and duplications or deletions of entire chromosomes. In the majority of cases, a direct causal link between a particular chromosomal rearrangement and a particular disease state has been found. This concomitant occurrence of disease with a chromosomal rearrangement can frequently be shown to result from alterations made in particular genes directly implicated in promoting the disease. [0004] The use of chromosome rearrangements as diagnostic and follow-up markers is based on several considerations. First, rearrangements are common in hematological malignancies and solid tumors and, as just indicated, the genes that are altered by the rearrangements are directly implicated in the development of malignancy. Second, rearrangements remain as clonal markers of the tumor cells throughout the progression of the disease. Third, rearrangements can be detected by cytogenetic or molecular methods. Examples of rearrangements that are currently useful in diagnostic, prognostic and follow-up evaluation of hematologic and solid tumor neoplasms are listed in Table 1 and Table 2 infra. [0005] Chromosome Translocations in Lymphomas. A number of chromosomal translocations have been identified as being correlated with certain lymphomas, including Non-Hodgkin's lymphoma (NHL). The incidence of NHL has increased at an alarming rate in recent years. It is now the fifth most common cancer in the U.S., and worldwide incidence is also increasing rapidly (Weisenburger 1994; Parker et al., 1996). Several translocations that correlate with various lymphoma subsets involve the IGH gene located on chromosome 14, at band q32 (14q32). Specific examples of such translocations include the t(8;14)(q24; q32) associated with Burkitt's Lymphoma (BL), the t(11;14)(q13; q32) in Mantle Cell Lymphoma (MCL), and t(14;18)(q32; q21) associated with follicular B-cell lymphoma (FL). [0006] The t(8;14)(q24; q32) translocation was the first recurring chromosomal translocation shown to be associated with lymphomas (Zech et al., 1976). Further studies established this translocation as the hallmark of Burkitt's lymphoma (BL) because 100% of the cases show this translocation; in addition, it also has been reported in about 15% of other high grade B-cell lymphomas. (Offit et al., 1991; 1992; Gaidano et al., 1993). [0007] MCL originates from the mantle zone B-cells. It is seen in 5-10% of NHL in adults (Weisenburger, 1992; Raffeld, et al., 1991; Banks, et al., 1992). The t(11;14)(q13; q32) is a consistent cytogenetic abnormality in 70% to 90% of MCL's (Tsujimoto et al., 1984; Williams et al., 1991; Coignet et al., 1996). The t(14;18)(q32; q21) translocation characterizes approximately 60% of B-cell NHL. It is seen in 85% of the FL and 25% of diffuse aggressive large cell lymphomas (DLCL). [0008] The physiologic consequence of each of these translocations is deregulation of expression of a gene located at the breakpoint of the chromosome band rearranging with 14q32. This deregulation is brought about by replacement of the gene's regulatory signals by those of the IGH gene, which are consistently expressed in B cells. Thus, MYC, BCL-1, and BCL-2 are the target deregulated genes in BL, MCL, and FL, respectively, associated translocations (Chaganti, et al., 2000). In addition to these common or well-known translocations, more than a dozen other less frequent, but equally important, translocations have been identified (Chaganti, et al., 2000). Because breakpoints of the translocations occur in circumscribed regions of chromosomal DNA, their detection can be of value in diagnosis and post therapy follow-up of these neoplasms. [0009] Methods for the Detection of Chromosomal Rearrangements. Several approaches to detecting chromosomal rearrangements have been developed. These include karyotype band analysis, Southern blot analysis, and analysis by quantitative polymerase chain reaction (PCR) or reverse-transcription PCR (RT-PCR). Of these various methods, karyotype analysis by conventional banding is the most accurate method currently in use. [0010] Karyotyping involves culturing tumor cells from bone marrow aspirate, lymph node, or other tissue biopsies. The methodology involves accumulating metaphase cells by treatment with colcemid and fixation on glass slides according to established preparative procedures. Slides containing adequate numbers of cells in metaphase-prometaphase stage are "banded" by a number of techniques, G-banding being the most widely utilized, and observed under a microscope. The dark and pale bands along the length of the chromosome are consistent and reproducible (banding pattern). Consequently, they serve as landmarks that are the basis for chromosome identification, as well as assignment of breakpoints at the sites of rearrangement. It is precise as long as dividing cells are available. The drawbacks of this method include that it requires dividing cells, which is labor intensive and time consuming, and the method does not detect small deletions important in evaluating both the disease and potential treatments. The current rate of successful cytogenetic analysis of newly diagnosed cases ranges from 60% to 80% for hematological malignancies and <40% for solid tumors. This percentage falls dramatically in post-treatment bone marrow/blood samples, making cytogenetics a less useful tool for patient follow-up. Fifty percent of follow-up specimens do not yield results due to the extreme hypocellularity of marrow samples and poor proliferation in vitro. [0011] Southern Blot analysis involves isolating DNA from a tumor sample and subjecting it to endonuclease digestion and fractionation on agarose gels using electrophoresis. The size-separated DNA fragments are then transferred to a nylon membrane and probed by hybridization with DNA sequences adjacent to the breakpoint. A novel rearranged band, in addition to the germ line band derived from the normal allele, indicate the presence of the translocation. This method requires the use of probes in the vicinity of the suspected breakpoints to detect rearrangements. Moreover, detection of rearrangements depends on clustering of the breakpoints in one of the rearranging chromosomes within a small region, which can be detected by one or few restriction enzyme digests. Detection of translocations, which have multiple breakpoints, requires multiple probes and multiple enzyme digests of DNA, which is cost inefficient. As a consequence, Southern blotting is not readily applicable for detecting all breakpoints in a number of disease-associated translocations [e.g., t(8;14)(q24q32), t(11;14)(q13q32) and t(14;18)(q32q21)]. Finally, Southern blotting does not possess the sensitivity required to detect translocations in heterogenous samples, as the method requires a sample comprised of at least,5-10% tumor cells carrying the chromosomal rearrangement. [0012] The polymerase chain reaction (PCR) relies on amplification of a PCR product from the translocation junction using primers containing sequences near the vicinity of the breakpoint derived from both chromosomes involved in the translocation. PCR amplification will occur only from tumor cells, whereas an amplification product will not be generated from the DNA of normal cells in which the primer templates remain on separate chromosomes. Genomic PCR will successfully amplify only if the breakpoints in the two chromosomes involved in the translocation in different tumors are distributed within a distance of 1 to 2 KB, which is the limit of conventional PCR amplification. [0013] Reverse transcription PCR (RT-PCR) is used to identify fusion RNA and can also be employed in cases with widely scattered breakpoints. As with conventional PCR, successful amplification depends on consistent breakpoints within the same intronic regions of the two genes. In the RT-PCR method, the primers are selected from the sequences of the exons near the breakpoints from each of the two genes involved in the translocation. Although both DNA and RT-PCR methods are sensitive enough to amplify DNA, they are difficult to quantify and do not provide information on disease in terms of proportion of cells in a given biopsy, which is the desired measure. In addition, necrotic cells or free DNA can lead to biologically questionable positive results. [0014] Both versions of the PCR analysis are difficult to quantitate and prone to false positive results from amplification of DNA sequences in dead tumor cells or in free DNA from lysed cells still present in the specimen. Moreover, PCR techniques are not readily applicable to detecting many disease-associated translocations because of the dispersed nature of the breakpoints found in these arrangements, with the dispersement of breakpoints necessitating multiple PCR reactions. These methods also have a limited utility because they are not applicable to many rearrangements involving deletions that are frequently associated with poor prognosis, such as those associated with the BCR/ABL regions on der (9) (Sinclair et al., 1999) and on der(22) (Palanisamy, unpublished data) which occur in cases of CML. [0015] Fluorescence in situ Hybridization (FISH) is another tool for detecting chromosomal rearrangements. The term "in situ hybridization" generally refers to hybridization of a nucleic acid probe to a nucleic acid target that is part of a cytological or histological preparation. Typically, FISH methods involve the following steps: (a) fixing the tissue or other biological material under investigation to a support (e.g., glass slide or wall of a micro titer well), (b) treatment of the tissue or material to increase accessibility of probe DNA to target DNA, (c) contacting the tissue or material containing the target DNA with probes to form specific hybridization complexes, (d) post hybridization washes of the complexes to selectively remove probes that are not specifically hybridized to the target, and (e) detection of probes that have formed hybridization complexes with target DNA molecules. An advantage of FISH is that one can analyze individual cells, which eliminates the need to utilize cycling cells. Such methods are described in a number of sources, including: Gall and Pardue, (1981) Methods of Enzymology 21:470-480; Henderson, (1982) International Review of Cytology, 76:1-46; and Angerer, et al., (1985) in Genetic Engineering: Principles and Methods (Setlow and Hollaender, Eds.) vol. 7, pp. 43-65, Plenum Press, New York. [0016] One of the problems with interphase FISH analysis is signal artifacts. These arise from two sources. One is non-specific binding of labeled DNA to protein in intact fixed nuclei. This can be partly overcome by treating the nuclei with proteolytic enzymes such as proteinase K or trypsin. Recent advances in imaging technology enables one to capture images using a CCD (Charge Coupled Device) camera and processing of images using image-processing software to reduce the background fluorescence arising from non-specific hybridization. Another problem is non-specific cross hybridization of repeat sequences in the probe with those in the genome. A Cot-1 DNA suppression step can be included in the hybridization protocols to ameliorate this problem. Because nucleic acids in the Cot-I fraction are characterized by containing highly repetitive sequences (e.g., Alu sequences, .alpha.-satellite, .beta.-satellite sequences), these nucleic acids bind to repeat sequences in the genome, thereby blocking binding of probes to such sequences (see, e.g., Benjamin Lewin, (1994) Genes V, Oxford University Press). The limitation of such approaches, however, is that such steps fail to bind all repeat sequences and fail to anneal to all the copies of any given sequence. [0017] Application of Detection Methods to Translocations Associated with Lymphomas. The heterogeneous nature of NHL makes accurate disease diagnosis and follow-up particularly challenging. Cytological and histological methods were previously the diagnostic methods of choice. Recent advances in developing immunophenotypic markers as well as molecular methods discussed supra, such as PCR, have revealed the highly heterogeneous nature of lymphoma. It is well established that each lymphoma type is characterized by a specific immunophenotype marker as well as nonrandom chromosomal abnormalities. [0018] NHLs are treated by a variety of chemotherapeutic regimens and/or autologous bone marrow transplantation, which require regular follow-up and monitoring for recurrence or cancer in bone marrow and blood. The gold standard currently in use for this purpose is karyotype analysis by G-banding. As indicated supra, although this method is precise, it is labor intensive; in addition, 50% of follow-up specimens do not yield results, due to extreme hypocellularity of marrow samples and poor in vitro proliferation. Due to the dispersed nature of the breakpoints in many translocations [e.g., t(8;14)(q24; q32), t(11;14)(q13; q32) and t(14;18)(q32; q21)] over a large genomic region and the requirement of multiple probes and multiple PCR reactions, Southern blotting and PCR methods are not readily applicable for detecting all breakpoints. Furthermore, Southern blotting and PCR methods are prone to sensitivity and specificity problems because, as noted above, Southern blotting requires the presence of at least 5-10% tumor cells in the sample to detect a rearrangement, and PCR is prone to generate false positive results due to amplification of DNA from dead tumor cells or lysed cells that may be present in the sample. [0019] FACS analysis can be used to detect specific combinations of cell surface proteins that may characterize the original blast (i.e., the immature stage of cellular development before appearance of the defining characteristics of the cell) population. Using FACS analysis, abnormal populations can be detected in as few as 0.1% (10-3) of cells. PCR has emerged as the most promising approach to date for the detection of minimum residual disease (MRD; This is the lowest percentage of tumor cells, within a large population of normal cells, at which the clinical condition of the patient can be said to be in remission. Thus, this is a symptom free disease condition, despite the presence of a low percentage of tumor cells), although other methods such as pathologic examination, FACS, PCR and cytogenetics are also utilized to estimate MRD. While PCR offers the advantage of very high sensitivity, with a detection limit of approximately 10.sup.-5, the problems noted above concerning lack of quantitation and generation of spurious results during the amplification process means that the technique is not particularly attractive for estimating MRD. [0020] Hence, all currently available rearrangement detection methods have various shortcomings. Consequently, new methods are needed to successfully monitor the presence of tumor cells to enable evaluation of treatment options and prediction of recurrence. SUMMARY [0021] A variety of methods and probes are provided herein for use in the detection of chromosomal rearrangements, including methods utilizing fluorescent in situ hybridization (FISH) methods. By utilizing various techniques that are disclosed herein either singularly or in combination, the methods and probes enhance the sensitivity and the specificity of rearrangement detection relative to other conventional approaches. [0022] Certain methods involve the preparation of probes that are free of repeat sequences and which are selected to hybridize to regions that flank potential breakpoints involved in the translocation of interest. Such methods typically involve a series of steps which include: (a) preparing a first collection of fragments that hybridize to regions flanking potential break points of a first chromosome involved in the chromosomal translocation; (b) preparing a second collection of fragments that hybridize to regions flanking potential break points of a second chromosome involved in the chromosomal translocation; (c) forming a first population of clones, each clone containing a fragment from the first collection linked to a vector; (d) forming a second population of clones, each clone containing a fragment from the second collection linked to a vector; (e) hybridizing fragments from the first population of clones with a sequence to allow elimination of clones containing fragments hybridizing to the repeat sequence; (f) hybridizing fragments from the second population of clones with a sequence to allow elimination of clones containing fragments hybridizing to the repeat sequence; (g) preparing probes from the clones in the first population whose vectors are linked to fragments that do not hybridize to the repeat sequence; and (h) preparing probes from the clones in the second population whose vectors are linked to fragments that do not hybridize to the repeat sequence. 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