METHODS FOR DETECTING TCR-GAMMA GENE REARRANGEMENT

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001 ] This application claims the benefit of U.S. Applications 61/ 140,029, filed December 22, 2008 and 12/491,170, filed June 24, 2009, each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] This invention relates to the field of cancer diagnosis. In particular, the invention relates to the determination of clonality of T-cell, diagnosis and prognosis of patients having lymphoproliferative disease associated with T-cell receptor gamma (TCR-γ) gene rearrangement.

BACKGROUND OF THE INVENTION

[0003] The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the invention.

[0004] T cell receptors recognize foreign antigens which have been processed as small peptides and bound to major histocompatibility complex (MHC) molecules at the surface of antigen presenting cells (APC). Each T cell receptor is a dimer consisting of one alpha and one beta chain or one delta and one gamma chain. The human T-cell receptor gamma (TCR-γ) locus is located on chromosome 7 (7p 14) and includes V (variable), J (joining), and C (constant) segments (Lcfranc ct al. Curr. Top. Microbiol. Immunol. 1991 ; 173: 3-9).

[0005] To date 14 V gene (V γ) segments (TRGVl , TRG V2, TRGV3, TRGV4, TRG V5, TRGV5P, TRGV6, TRGV7, TRGV8, TRGVA, TRGV9, TRGVl O, TRGVB, TRGVl 1), including 6 pseudogenes have been identified upstream to five J gene (J γ) segments (TRGJPl , TRGJP, TRGJl , TRGJP2, and TRGJ2), and two C gene (C γ) segments (TRGCl and TRGC2) in the TCR-γ locus (Lefranc et al. Nature. 1985; 316: 464-466; Lefranc et al. Cell. 1986; 45: 237-246; Quertermous et al. Science. 1986; 231 : 252-255; Forster et al. EMBO J. 1987; 6: 1945-1950; Huck et al. EMBO J. 1988; 7(3): 719-726). Several V segments of the gamma locus are known to be incapable of encoding a protein and are considered pseudogenes.

[0006] Rearrangement of TCR-γ genes is an important part of thymocyte development. During T cell development, the gamma chain is synthesized by a recombination event at the DNA level joining a V segment with a J segment; the C segment is later joined by splicing at the RNA level. Recombination of many different V segments with several J segments provides a wide range of antigen recognition. The status of TCR-γ gene rearrangements helps define developmental stages. Analysis of TCR-γ gene rearrangement can be used to detect clonality in a T-cell population (Signoretti et al. Am. J. Pathol. 1999; 154: 67-75; Chain et al. J. Immunol. Methods. 2005; 300(1-2): 12-23).

[0007] Clonality is not synonymous with malignancy because it can be detected in nonneoplastic lymphocytic infiltrates (Wood et al. J Invest Dermatol. 1994; 103: 34-41). Nevertheless, it is generally accepted that most neoplasms are clonal in origin. Thus, detection of clonal cells with identical rearrangement favors a diagnosis of malignancy. Peripheral T-cell lymphomas arise from T cells that undergo malignant transformation after most rearrangements of TCR loci are completed. T-cell clonality estimation is important for the differential diagnosis between malignant and nonmalignant T-cell proliferation (Diss et al. J Clin Pathol. 1995; 48: 1045-1050; Signoretti et al. Am. J. Pathol. 1999; 154: 67-75; Gra et al. J. MoI. Diagn. 2007; 9: 249-257).

SUMMARY OF THE INVENTION

[0008] This invention relates to detection of TCR-γ nucleic acid in acellular body fluid. The invention is useful for detecting TCR-γ gene rearrangements in such fluid and for diagnosis relating to lymphoproliferative disorder.

[0009] In one aspect, the invention provides a method for diagnosing an individual as having a lymphoproliferative disorder, the method comprising: a) providing an acellular bodily fluid sample from the individual, wherein the sample comprises TCR-γ nucleic acid: b) determining the relative abundance of a plurality of V-γ/J-γ gene rearrangements in the TCR-γ nucleic acid; and d) identifying the individual as having a lymphoproliferative disorder when the TCR-γ nucleic acid comprises at least one major arrangement.

[0010] In one aspect, the invention provides a method for identifying the rearrangement of the TCR-γ gene in an individual. The method includes a) providing an acellular bodily fluid sample of the individual comprising TCR-γ nucleic acid b) identifying the arrangement of V γ gene segments joined with J γ gene segments on a single polynucleotide. The arrangement of V γ joined with J γ gene segments on a single polynucleotide in the acellular body fluid is indicative of rearrangement of the TCR-γ gene.

[0011] In another aspect, the invention provides a method of determining the clonality of a T-cell population of an individual comprising: evaluating nucleic acid from an acellular bodily fluid sample of the individual to determine the relative abundance of a plurality of V- γ/J-γ gene rearrangements in the TCR-γ nucleic acid, and identifying the individual as having a monoclonal T-cell population when at least one major arrangement is identified. The presence of a multiple rearrangements of the V γ and J γ gene segments in TCR-γ gene is indicative of polyclonality.

[0012] In some embodiments of the above aspects of the invention, the relative abundance of a plurality of V-γ/J-γ gene rearrangements in the TCR-γ nucleic acid in the sample may be compared with the relative abundance of a plurality of V-γ/J-γ gene rearrangements of a reference TCR γ nucleic acid. In some embodiments, the reference TCR γ nucleic acid may be derived from polyclonal T-cells. In another embodiment, the reference TCR γ nucleic acid may be derived from monoclonal T-cells. In one embodiment of the above aspects of the invention, the method further includes identifying at least one major arrangement and comparing the identity of at least one major arrangement to known V- γ/J— γ gene rearrangements.

[0013] In some embodiments of the above aspects of the invention, major V-γ/J-γ gene rearrangements comprise at least 10%, at least 25%, at least 50% or at least 80% of the TCR- γ nucleic acid present in the acellular body fluid sample. [0014] In certain embodiments of these methods, the TCR-γ gene arrangement is determined by amplifying at least a portion of the TCR-γ nucleic acid, for example, using one or more oligonucleotide primers directed to any one or more (e.g., two, three, four, five, six, or more) of the V-γ gene segments and/or any one or more (e.g., two, three, four, five, six, or more) of the J-γ gene segments. In other embodiments, the TCR-γ gene arrangement is any one or more of those identified in Table 1. Suitable amplification primers include, for example, oligonucleotide primers containing the sequence of SEQ ID No's: 2 and/or 3, or complements thereof. In some embodiments, the amplification method may also include a third and/or a fourth primer (e.g., nested PCR or semi-nested PCR). The TCR-γ gene arrangement, including the identity of the V-γ and J-γ gene segments, may be determined using any suitable method including, for example, by determining the nucleotide sequence of all or a portion (e.g., the V-γ and J-γ gene segment junction) of the TCR-γ nucleic acid, by oligonucleotide probe hybridization. In one embodiment, the identity of at least one major arrangement is determined using a nucleic acid probe specific for the junction of a portion of V-γ gene segments and a portion of J-γ gene segment. Optionally, amplified TCR-γ nucleic acid or portion thereof comprising one or more V-γ/ J-γ gene rearrangements may be separated by size. The size of the amplified TCR-γ nucleic acid may be determined by various techniques known in the art such as, gel electrophoresis, chromatography (e.g., capillary electrophoresis, HPLC, size exclusion chromatography).

[0015] In some embodiments, the TCR-γ gene rearrangement may be detected by Southern blot. In another embodiment, the TCR-γ gene rearrangement may be detected by fluorescent in situ hybridization, by flow cytometry, by hybridization of a probe to genomic DNA comprising the rearrangement.

[0016] In one embodiment, the TCR-γ gene rearrangement may be detected by real time PCR using TaqMan® probes, In one embodiment, the primers used to detect the TCR-γ gene rearrangement in a real time PCR reaction may be SEQ ID NO: 2 and SEQ ID NO: 3. In other embodiments, the forward primers may be any of SEQ ID NO: 4-6, and 10-13 and reverse primers may be any of SEQ ID NO: 7-9, 14-16. In one embodiment, the TaqMan® probe used to detect the TCR-γ gene rearrangement in a real time PCR reaction may contain SEQ ID NO: 17-29. [0017] In some embodiments, the nucleic acid of a second gene is amplified from the acellular body fluid as an internal control The second gene is preferably one which is unrelated to TCR-γ cither structurally or functionally and is not associated with a lymphoproliferatrv e disorder

[0018] In other embodiments, the identity of the TCR-γ gene arrangement is used to identify a specific lymphoproliferative disorder such as, for example, T-cell acute lymphoblastic leukemia, large granular lymphocytic leukemia, mycosis fungoides, peripheral T-cell lymphoma, T-lymphoblastic lymphoma, T-cell prolymphocyte leukemia, Sezary syndrome, and γδ-hepatosplemc lymphoma In other embodiments, the lymphoproliferative disorder is diagnosed by identifying the presence of a clonal T cell population expressing any one of the TCR-γ gene arrangement identified m Table 1

[0019] "Individual" as used herein means a human or any other animal which contains a TCR-γ nucleic acid that can be amplified using the primers and methods described herein An individual can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease A human includes pre and post natal forms

[0020] "Sample" or "patient sample" as used herein includes biological samples such as tissues and body fluids "Body fluids" may include, but are not limited to, blood, serum, plasma, saliva, cerebral spinal fluid, pleural fluid, tears, lactal duct fluid, lymph, sputum, uπne, amniotic fluid, and semen A sample may include a body fluid that is "acellular " An "acellular body fluid" includes less than about 1% (w/w) whole cellular mateπal Plasma or serums are examples of acellular bodily fluids An acellular sample can be deπved from a sample compπsing cells by removing more than 99% (w/w) of the cells from sample A sample may include a specimen of natural or synthetic origin

[0021] "Plasma" as used herein refers to acellular body fluid deπved from blood by removing whole cellular mateπal from blood by methods known in the art (e g , centπlugation, filtration, and the like)

[0022] "Serum" is an acellular body fluid obtained from clotted blood after the clotted fraction is removed [0023] "Nucleic acid" or "nucleic acid sequence" as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, which may be single or double stranded, and represent the sense or antisense strand. A nucleic acid may include DNA or RNA, and may be of natural or synthetic origin and may contain deoxyribonucleotides, ribonucleotides, or nucleotide analogs in any combination.

[0024] Non-limiting examples of polynucleotides include a gene or gene fragment, genomic DNA, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, synthetic nucleic acid, nucleic acid probes and primers. Polynucleotides may be natural or synthetic. Polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches. A nucleic acid may be modified such as by conjugation, with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of chemical entities for attaching the polynucleotide to other molecules such as proteins, metal ions, labeling components, other polynucleotides or a solid support. Nucleic acid may include nucleic acid that has been amplified (e.g., using polymerase chain reaction).

[0025] "Genomic nucleic acid" as used herein refers to the nucleic acid in a cell that is present in the cell chromosome(s) of an organism which contains the genes that encode the various proteins of the cells of that organism. A preferred type of genomic nucleic acid is that present in the nucleus of a eukaryotic cell. In a preferred embodiment a genomic nucleic acid is DNA. Genomic nucleic acid can be double stranded or single stranded, or partially double stranded, or partially single stranded or a hairpin molecule. Genomic nucleic acid may be intact or fragmented (e.g., digested with restriction endonucleases or by sυnication or by applying shearing force by methods known in the art). In some cases, genomic nucleic acid may include sequence from all or a portion of a single gene or from multiple genes, sequence from one or more chromosomes, or sequence from all chromosomes of a cell. As is well known, genomic nucleic acid includes gene coding regions, introns, 5' and 3' untranslated regions, 5' and 3' flanking DNA and structural segments such as tclomeric and centromeric DNA, replication origins, and intergenic DNA. Genomic nucleic acid representing the total nucleic acid of the genome is referred to as "total genomic nucleic acid "

[0026] Genomic nucleic acid may be obtained by methods of extraction/purification from acellular body fluids as is well known in the art The ultimate source of genomic nucleic acid can be normal cells or may be cells that contain one or more mutations in the genomic nucleic acid, e g , duplication, deletion, translocation, and transversion Included in the meaning of genomic nucleic acid is genomic nucleic acid that has undergone recombination and may comprise rearranged genes Also included in the meaning of genomic nucleic acid is genomic nucleic acid that has been subjected to an amplification step that increases the amount of the target sequence of interest sought to be detected relative to other nucleic acid sequences in the genomic nucleic acid

[0027] "Gene" as used herein refers to a DNA sequence that comprises control and coding sequences necessary for the production of an RNA, which may have a non-coding function (e g , a πbosomal or transfer RNA) or which may include a polypeptide or a polypeptide precursor The RNA or polypeptide may be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained Included in the meaning of genes that do not encode a functional protein Such genes are often referred to as pseudogenes

[0028] The term "reference TCR-γ nucleic acid" as used herein means TCR-γ nucleic acid where the arrangement of variable (V) and joining (J) gene segments are known or the source of TCR-γ nucleic acid is known A reference TCR-γ nucleic acid can be deπved from monoclonal T-cclls A reference TCR-γ nucleic acid can be derived from polyclonal T-cells A reference TCR-γ nucleic acid may be a TCR-γ nucleic acid with known rearranged vanable (V) and joining (J) gene segments A reference TCR-γ nucleic acid can be TCR-γ genomic nucleic acid where variable (V) and joining (J) gene segments have not been rearranged such as the nucleic acid sequence of SFQ ID NO 1

[0029] The term "rearranged" or "rearrangement" as used herein in the context of TCR-γ gene means a nucleic acid sequence or an event where V and J gene segments of TCR-γ have been rearranged with respect to the reference TCR-γ nucleic acid sequence such as SEQ ID NO: 1. A V-J rearrangement may result from genetic recombination.

[0030] The term "major arrangement" as used herein in the context of the arrangement of V-γ and J-γ gene segment of TCR-γ gene means that the arrangement is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 99% or 100% of total possible arrangements of V-γ and J-γ gene segments of TCR-γ gene present in a acellular body fluid sample,

[0031 ] The term "relative abundance" as used herein in the context of the arrangement of V-γ and J-γ gene segment of TCR-γ gene means the proportion of an arrangement of V-γ and J-γ gene segment relative to the total arrangements of V-γ and J-γ gene segments in TCR-γ gene present in a nucleic acid sample,

[0032] The term "V-γ/J-γ gene rearrangements" as used herein means the arrangements of V-γ gene segments joined with J-γ gene segments in single TCR-γ nucleic acid,

[0033] The term "clonality" or "clonal" as used herein in the context of T-cell means how the T-cells are derived from a source. For example, monoclonal T-cells were derived from one cell type having one specific arrangement of V and J gene segments. On the other hand, polyclonal T-cells were derived from more than one cell type having more than one arrangement of V and J gene segments.

[0034] "Identity" and "identical" as used herein refer to a degree of identity between sequences. There may be partial identity or complete identity, A partially identical sequence is one that is less than 100% identical to another sequence. Preferably, partially identical sequences have an overall identity of at least 70% or at least 75%, more preferably at least 80% or at least 85%, most preferably at least 90% or at least 95% or at least 99%. Sequence identity determinations may be made for sequences which arc not fully aligned. In such instances, the most related segments may be aligned for optimal sequence identity by and the overall sequence identity reduced by a penalty for gaps in the alignment. [0035] "Substantially all" as used herein means at least about 60%, 70%, 80%, 90%, or 95-100%.

[0036] "Substantially pure" as used herein means a nucleic acid, represents more than 50%, 60%, 75%, 90%, 95% or more of the nucleic acid in a sample. The nucleic acid sample may exist in solution or as a dry preparation.

[0037] "Isolated" as used herein when referring to a nucleic acid (e.g., an oligonucleotide such as RNA, DNA, or a mixed polymer) means a nucleic acid that is apart from a substantial portion of the genome in which it naturally occurs and/or is substantially separated from other cellular components which naturally accompany such nucleic acid. For example, any nucleic acid that has been produced synthetically (e.g., by serial base condensation) is considered to be isolated. Likewise, nucleic acids that are recombinantly expressed, cloned, produced by a primer extension reaction (e.g., PCR), or otherwise excised from a genome are also considered to be isolated.

[0038] "A portion of in the context of a nucleic acid refers to a sequence of nucleotide residues which are at least about 10 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 100 nucleotides, at least about 250 nucleotides, at least about 500 nucleotides, at least about 1 ,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 10,000 nucleotides, at least about 20,000 nucleotides, at least about 50,000 nucleotides, at least about 100,000 nucleotides, at least about 500,000 nucleotides, at least about 1,000,000 nucleotides or more.

[0039] "Specific hybridization" as used herein is an indication that two nucleic acid sequences hybridize to each other but not to other related sequences under chosen hybridization conditions. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after any subsequent washing steps. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may occur, for example, at 65 ⁰ C in the presence of about 6><SSC. Stringency of hybridization may be expressed, in part, with reference to the temperature under which the wash steps are carried out. Such temperatures are typically selected to be about 5 ⁰ C to 20 ⁰ C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Equations for calculating Tm and conditions for nucleic acid hybridization are known in the art.

[0040J "High stringency hybridization conditions" as used herein refers to hybridization under conditions at least as stringent as the following: hybridization in 50% formamide, 5x SSC, 50 mM NaH ₂ PO ₄ , pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5x Denhart's solution at 42" C. overnight; washing with 2x SSC, 0.1 % SDS at 45° C; and washing with 0,2x SSC, 0.1% SDS at 45° C.

[0041] "Complement" as used herein refers to the complementary sequence to a nucleic acid according to standard Watson'Crick pairing rules. A complement sequence can also be a sequence of RNA complementary to the DNA sequence or its complement sequence, and can also be a cDNA. The complement of a nucleic acid sequence as used herein refers to an polynucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in "antiparallel association". For example, for the sequence "5'-A-G-T-3"' is complementary to the sequence "3'-T-C-A-5". Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs.

[0042] "Substantially complementary" as used herein means that two sequences hybridize under highly stringent hybridization conditions. The skilled artisan will understand that substantially complementary sequences need not hybridize along their entire length.

[0043] "Oligonucleotide" as used herein refers to a molecule that has a sequence of nucleic acid bases on a backbone comprised mainly of identical monomer units at defined intervals. The bases are arranged on the backbone in such a way that they can enter into a bond with a nucleic acid having a sequence of bases that are complementary to the bases of the oligonucleotide. The most common oligonucleotides have a backbone of sugar phosphate units. A distinction may be made betw een ohgodeoxyπbonucleotides that do not have a hydroxyl group at the 2' position and oligoribonucleotides that have a hydroxyl group in this position. Oligonucleotides also may include derivatives, in which the hydrogen of the hydroxyl group is replaced with organic groups, e.g., an allyl group. Oligonucleotides may compπse modified nucleobases, modified sugar moieties, and modified mternucleotide linkages.

[0044] Oligonucleotides can be used as primers or probes for specifically amplifying (i e , amplifying a particular target nucleic acid sequence) or specifically detecting (i e , detecting a particular target nucleic acid sequence) a target nucleic acid and generally are capable of specifically hybridizing to the target nucleic acid Oligonucleotides of the method which function as pnmers or probes are generally at least about 10-15 nucleotides long and more preferably at least about 15 to 25 nucleotides long, although shorter or longer oligonucleotides may be used in the method. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide The oligonucleotide may be generated in any manner, including, for example, chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof The oligonucleotide may be modified. For example, the oligonucleotide may be labeled with an agent that produces a detectable signal (e.g., a fluorophore).

[0045] "Primer" as used herein refers to an oligonucleotide that is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated (e.g., primer extension associated with an application such as PCR) The primer is complementary to a target nucleotide sequence and it hybridizes to a substantially complementary sequence in the target and leads to addition of nucleotides to the 3 '-end of the primer in the presence of a DNA or RNA polymerase The 3 '- nucleotide of the primer should generally be complementary to the target sequence at a corresponding nucleotide position for optimal expression and amplification An oligonucleotide "primer" may occur naturally, as in a purified restriction digest or may be produced synthetically. The term "primer" as used herein includes all forms of pnmers that may be synthesized including peptide nucleic acid primers, locked nucleic acid primers, phosphorothioatc modified primers, labeled pnmers, and the like. Pnmers may comprise modified nucleobases, modified sugar moieties, and modified mternucleotide linkages [0046] Primers are typically between about 10 and about 100 nucleotides in length, and preferably at least about 10, 15, 20, 25, 30, 35, 40, 50. 60, 75, 100 nucleotides or more in length.. An optimal length for a particular primer application may be readily determined in the manner described in H. Erlich, PCR Technology, Principles and Application for DNA Amplification (1989).

[0047] "Probe" as used herein refers to nucleic acid that interacts with a target nucleic acid via hybridization. A probe may be fully complementary to a target nucleic acid sequence or partially complementary. The level of complementarity will depend on many factors based, in general, on the function of the probe. A probe or probes can be used, for example to detect the presence or absence of a mutation in a nucleic acid sequence by virtue of the sequence characteristics of the target. Probes can be labeled or unlabeled, or modified in any of a number of ways well known in the art. A probe may specifically hybridize to a target nucleic acid.

[0048] Probes may be DNA, RNA or a RNA/DNA hybrid. Probes may be oligonucleotides, artificial chromosomes, fragmented artificial chromosome, genomic nucleic acid, fragmented genomic nucleic acid, RNA, recombinant nucleic acid, fragmented recombinant nucleic acid, peptide nucleic acid (PNA), locked nucleic acid, oligomer of cyclic heterocycles, or conjugates of nucleic acid. Probes may comprise modified nucleobases, modified sugar moieties, and modified internucleotide linkages. A probe may be fully complementary to a target nucleic acid sequence or partially complementary. A probe may be used to detect the presence or absence of a target nucleic acid. Probes are typically at least about 10, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100 nucleotides or more in length. However, longer probes are possible. Longer probes can be from few hundred bases to few million bases in length (e.g. BAC, YACs, genomic DNA fragments etc.).

[0049] The term "detectable label" as used herein refers to a molecule or a compound or a group of molecules or a group of compounds associated with a nucleic acid and is used to identify the nucleic acid.

[0050] In some cases, the detectable label may be detected directly, In other cases, the detectable label may be a part of a binding pair, which can then be subsequently detected. Signals from the detectable label may be detected by various means and will depend on the nature of the detectable label Examples of means to detect detectable label include but are not limited to spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemilummescence, or an> other appropπate means

[0051 ] " I arget nucleic acid" as used herein refers to a nucleic acid molecule (e g DNA or RNA) containing a sequence that has at least partial complementaπty with a pπmer oligonucleotide and/or a probe oligonucleotide A probe may specifically hybridize to a target nucleic acid

[0052] "Detecting" as used herein in context of detecting a signal from a detectable label to indicate the presence of a nucleic acid of interest in the sample (or the presence or absence of a protein of interest in the sample) does not require the method to provide 100% sensitivity and/or 100% specificity As is well known, "sensitivity" is the probability that a test is positive, given that the person has a genomic nucleic acid sequence, while "specificity" is the probability that a test is negative, given that the person does not have the genomic nucleic acid sequence A sensitivity of at least 50% is preferred, although sensitivities of at least 60%, at least 70%, at least 80%, at least 90% and at least 99% are clearly more preferred A specificity of at least 50% is preferred, although specificity of at least 60%, at least 70%, at least 80%, at least 90% and at least 99% are clearly more preferred Detecting also encompasses assays with false positives and false negatives False negative rates may be 1%, 5%, 10%, 15%, 20% or even higher False positive rates may be 1%, 5%, 10%, 15%, 20% or even higher

[0053] "About" as used herein means in quantitative terms, plus or minus 10%

[0054] The phrase "lymphoproliferative disorders" refer to several disorders or several conditions in which lymphocytes are produced in excessne quantities Lymphoproliferative disorders may be malignant or non-malignant Non-limiting examples of such disorders include chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia, lymphomas, multiple myeloma, Wiskott-Aldrich syndrome, post-transplant lymphoproliferative disorder, Autoimmune lymphoproliferative syndrome (ALPS), lymphoid interstitial pneumonia, T cell acute lymphoblastic leukemia (ALL), large granular lymphocytic leukemia, peripheral T-cell lymphoma, T-lymphoblastic lymphoma, T-cell prolymphocyte leukemia, γδ-hepatosplenic lymphoma, Sezary syndrome, mycosis lungoides

[0055] "Diagnose" or "diagnosis" or "diagnosing" as used herein refer to distinguishing or identifying a disease, syndrome or condition or distinguishing or identifying a person having a particular disease, syndrome or condition Usually, a diagnosis of a disease or disorder is based on the evaluation of one or more factors and/oi symptoms that are indicative of the disease That is, a diagnosis can be made based on the presence, absence or amount of a factor which is indicative of presence or absence of the disease or condition Each factor or symptom that is considered to be indicative for the diagnosis of a particular disease does not need be exclusively related to the particular disease, i e there may be differential diagnoses that can be inferred from a diagnostic factor or symptom Likewise, there may be instances where a factor or symptom that is indicative of a particular disease is present in an individual that does not have the particular disease

[0056] "Treatment," "treating," or "treat" as used herein refers to care by procedures or application that are intended to rehe\e illness or injury Although it is preferred that treating a condition or disease will result in an improvement of the condition, the term treating as used herein does not indicate, imply, or require that the procedures or applications are at all successful in ameliorating symptoms associated with any particular condition Treating a patient may result in adverse side effects or even a worsening of the condition which the treatment was intended to improve

[0057] "Determining a prognosis" as used herein refers to the process in which the course or outcome of a condition in a patient is predicted The term "prognosis" docs not refer to the ability to predict the course or outcome of a condition with 100% accuracy Instead, the teπn refers to identifying an increased or decreased probability that a certain course or outcome will occur in a patient exhibiting a given condition'marker, when compared to those individuals not exhibiting the condition The nature of the prognosis is dependent upon the specific disease and the condition/marker being assessed For example, a prognosis may be expressed as the amount of time a patient can be expected to survive, the likelihood that the disease goes into remission, or to the amount of time the disease can be expected to remain in remission BRIEF DESCRIPTION OF THE FIGURES

[0058] FIG. 1 shows a schematic representation of reference TCR-γ genomic nucleic acid. Fig. IA shows the organization of the different gene segments in the reference TCR-γ genomic nucleic acid. Variable gene segments Vl-VI l, V5P, VA, VB are shown as open boxes. The variable region subgroups Vγ-I, Vγ-II, Vγ-III, and Vγ-IV are indicated. Joining gene segments JPl , JP, Jl, JP2, and J2 are shown as shaded boxes. Constant gene segments Cl and C2 are shown as solid boxes. Fig. IB shows exemplary rearrangements of TCR-γ genes. The positions of the primer that allow the amplification of the rearranged genes are indicated as arrows above the variable (V) and joining (J) gene segments. "N" represents variable diversity region at the junction of V-J segments.

[0059] FIG. 2 shows the results of the analysis of the PCR amplified fragments of IgH (FTG. 2A) and TCR-γ gene (FIG. 2B) by capillary gel electrophoresis (CGE). Representative CGE profiles of matched peripheral blood and plasma samples are shown. The expected product sizes ranged from 220-310 bp for IgH and 140-180 bp for TCR-γ.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention provides methods for detection of TCR-γ nucleic acid in acellular body fluid. Exemplary TCR-γ nucleic acid includes but is not limited to genomic DNA, mRNA, and cDNA derived from mRNA. The methods can be used to detect the rearrangement of TCR-γ genes. The status of TCR-γ gene rearrangements can help define the developmental stages and determine the clonality of T-cells. The methods of the present invention is useful in diagnosis of lymphoproliferative disorder. The methods are specially useful when it is difficult to diagnose a T-cell lymphoproliferative disorder based only on clinical, histological and immunohistochemical criteria.

[0061] TCR-γ nucleic acid

[0062] TCR-γ nucleic acid detected by the methods of the invention may be intact or fragmented and also may be double or single stranded. In some cases, TCR-γ nucleic acid is partially double stranded. Tn other cases, TCR-γ nucleic acid comprises a translated region. In yet other cases, TCR-γ nucleic acid may include an untranslated region. Non-limiting examples of untranslated region include introns and pseudogenes,

[0063] In some instances, TCR-γ nucleic acid may comprise TCR-γ reference nucleic acid. Reference TCR-γ nucleic acid may comprise variable gene V γ segments, joining gene (J γ) segments, and constant gene (C γ) segments. In some cases, the TCR-γ reference nucleic acid may be TCR-γ reference genomic nucleic acid nucleic acid such as SEQ ID NO: 1. A schematic representation of the organization of the V γ, J γ, and C γ gene segments in a reference TCR-γ genomic nucleic acid is shown in Fig. 1. In some cases, TCR-γ nucleic acid may comprise rearranged TCR-γ genes. In some cases, TCR-γ nucleic acid includes both reference TCR-γ nucleic acid and TCR-γ nucleic acid with rearranged TCR-γ genes.

[0064] Human TCR-γ locus (also known as TRG@, TRG, TCRG ) is located in human chromosome 7pl 4. Exemplary sequences of human chromosome 7 include but arc not limited to GenBank Accession numbers: NWJ)Ol 839003, NT_079596, NT_079592, NW 923240, NT__007819, NT 007914. Exemplary TCR-γ genomic nucleic acid includes but is not limited to GenBank Accession numbers NG__001336. Sequence of reference TCR-γ genomic nucleic acid is provided as SEQ ID NO: 1. These sequences are incorporated herein by reference.

[0065] Variable gene segments of TCR-γ locus As of date, 14 variable gene (V γ) segments in TCR-γ genomic nucleic acid are known and they include TRGVl , TRGV2, TRGV3, TRGV4, TRGV5, TRGV5P, TRGV6, TRGV7, TRGV8, TRGVA, TRGV9, TRGVlO, TRGVB, TRGVl 1 (Forster et al. EMBO J. 1987; 6: 1945-1950; Hara et al. J. Clin. Invest. 1989; 83: 1277-1283). Of these 14 variable genes, several V segments of the gamma locus are known to be incapable of encoding a protein and are considered pseudogenes. These pseudogenes include TRGVl , TRGV5P, TRGV6, TRGV7, TRGVA, TRGVlO, TRGVB.

[0066] Non-limiting exemplary sequences of variable gene (V γ) segments in TCR-γ locus are provided below. For TRGVl : nucleotides 5893-6362 of SEQ ID NO: 1, GenBank Accession numbers AH003002, Ml 2949; for TRGV2: nucleotides 10620-1 1084 of SEQ ID NO: 1 , GenBank Accession number Ml 3429; for TRGV3: nucleotides 14969-15436 of SEQ ID NO: 1 , GenBank Accession number Ml 3430; for TRG V4: nucleotides 19765-120233 of SEQ ID NO: 1 , GenBank Accession numbers Ml 3584, X 13354; for TRGV5: nucleotides 24124-24593 of SEQ ID NO: 1 , GenBank Accession number Xl 3355; for TRGV5P: nucleotides 28449-28918 of SEQ ID NO: 1 , GenBank Accession numbers M30893, Ml 3431 ; for TRGV6: nucleotides 32777-33248 of SEQ ID NO: 1, GenBank Accession number Ml 3432; for TRGV7: nucleotides 38434-38907 of SEQ ID NO: 1 , GenBank Accession number M 13433; for TRGV8: nucleotides 43127-43605 of SEQ ID NO: 1 , GenBank Accession number Ml 3434; for TRGVA: nucleotides 51085-51502 of SEQ ID NO: 1 ; for TRGV9: nucleotides 56456-56931 of SEQ ID NO: 1, GenBank Accession number Xl 5274; for TRGVlO: nucleotides 73668-74138 of SEQ ID NO: 1, GenBank Accession number X07206, for TRGVB: nucleotides 77715-78185 of SEQ ID NO: 1 ; for TRGVI l : nucleotides 81870-82332 of SEQ ID NO: 1 , GenBank Accession number X07207. Sequences of the

V γ segments listed in the GenBank Accession numbers indicated above are incorporated herein by reference.

[0067] Based on the sequence homology the V γ segments are categorized into 4 families

V γ -I (TRGVl , TRGV2, TRGV3, TRGV4, TRGV5, TRGV5P, TRGV6, TRGV7, TRGV8); V γ -II (TRGV9); V γ -III (TRGVl O) and V γ -IV (TRGVI l) (LeFranc et al. Cell. 1986; 45: 237-246; Huck et al. FEBS Lett. 1987; 224: 291 296; Theodorou et al. J Pathol. 1994; 174: 233^242). Members of V γ -I family of genes have closely related sequences.

[0068] Joining gene segments of TCR-γ locus Non-limiting exemplary sequences of the joining gene (J γ) segments are provided below. For TRGJPl : nucleotides 97630-97689 of SEQ ID NO: 1 , GenBank Accession number X08084; for TRGJP: nucleotides 100300- 100361 of SEQ ID NO: 1. GenBank Accession number X58182, M12950; for TRGJl : nucleotides 104408-104457 of SEQ ID NO: 1, GenBank Accession number M12960; for TRGJP2: nucleotides 1 17552-11761 1 of SEQ ID NO: 1 , GenBank Accession number Ml 6016; for TRGJ2: nucleotides 120519-120568 of SEQ ID NO: 1, GenBank Accession number M12961. Sequences of the J γ segments listed in the GenBank Accession numbers indicated above are incorporated herein by reference.

[0069] Constant gene segments of TCR-γ locus Two constant gene (C γ) segments TRGCl and TRGC2 for TCR-γ locus are known. Non-limiting exemplary sequences of the constant gene (C γ) segments are provided below For TRGCl nucleotides 108270-1 13860 of SEQ ID NO 1 and for TRGC2 nucleotides 120519-120568 of SEQ ID NO 1

[0070] Diversity regions In some embodiments, nucleic acid compπsing the junction ot

V γ -J γ segments may further compπse a diversity region (N) with sequences of variable length and are described in for example, Huck et al EMBO J 1988, 7(3) 719-726 Sequences of these diversity regions (N) are incorporated herein by reference

[0071 ] Rearrangement of TCR-γ genes

[0072] Rearrangement of TCR-γ genes is an important part of thymocyte dev elopment Duπng T cell development, the gamma chain is synthesized by a recombination event at the DNA level joining a V segment with a J segment, the C segment is later joined by splicing at the RNA level Recombination of many different V segments with several J segments provides a wide range of antigen recognition

[0073] Analysis of TCR-γ gene rearrangement can be used to detect clonality in a T-cell population (Signoretti et al Am J Pathol 1999, 154 67-75, Cham et al J Immunol Methods 2005, 300(1-2) 12-23) Clonality is not synonymous with malignancy because it can be detected in nonneoplastic lymphocytic infiltrates (Wood et al J Invest Dermatol 1994, 103 34-41) Nevertheless, it is generally accepted that most neoplasms are clonal in oπgin Thus, detection of clonal cells with identical rearrangement favors a diagnosis of malignancy Peπpheral T-cell lymphomas arise from T cells that undergo malignant transformation after most rearrangements of TCR loci are completed T-cell clonality estimation is important for the differential diagnosis between malignant and nonmahgnant T- cell proliferation (Diss et al J Clin Pathol 1995, 48 1045-1050, Signoretti et al Am J Pathol 1999, 154 67-75, Gra et al J MoI Diagn 2007, 9 249-257)

[0074] Regarding the variable genes in the TCR-γ -rearranged alleles, members of the

V γ -I subgroup were the most frequently used, followed by ^" V γ -II, V γ -III, and V γ -IV Joining segment usage was as follows Jl or J2 are used more often than JPl or JP2 ( lhcodorou ct al J Pathol 1994, 174 233-242) [OO75J The analysis of TCR-γ gene rearrangement is informative about the T-cell clonality. TCR-γ gene rearrangement is often associated lymphoproliferative disorder. Thus determination of T-cell clonality by assessing the TCR-γ gene rearrangement is useful in the diagnosis of lymphoproliferative disorder. The determination of T-ccll clonality by assessing the TCR-γ gene rearrangement is specially useful when diagnosis is difficult based on clinical, histological and immunohistochemical criteria.

[0076] Exemplary TCR-γ gene rearrangements associated with several lymphoproliferative disorders are listed in Table 1 below. Although the listed TCR-γ gene rearrangements are commonly associated with lymphoproliferative disorder, other TCR-γ gene rearrangements are possible. Any of such rearrangements can be detected by the methods of the present invention.

TABLE 1 : Exemplary TCR-γ gene rearrangements associated with several lymphoproliferative disorders

LGL: large granular lymphocytic leukemia, MF mycosis fungoides: , PTCL: peripheral T-cell lymphoma, TLL: T-lymphoblastic lymphoma, TPLL: T-cell prolymphocytic leukemia, SS: Sezary syndrome, HSL: γδ-hepatosplenic lymphoma. Exemplary rearrangement indicated as (TRG V4, TRGV9, TRGJ 1/TRGJ2) means gene segments TRGV4 (Vγ4) is joined to TRG V9 (Vγ9), and TRGV9 is joined to TRGJl (Jγl) or TRGJ2 (Jγ2) by recombination resulting in a nucleic acid sequence Vγ4-Vγ9-Jγl or Vγ4-Vγ9-Jγl on a single polynucleotide.

[0077] TCR-γ mRNA Full length and partial sequences of rearranged TCR-γ mRNA sequences are known. Exemplary sequences includes GenBank accession numbers: M30892, M30891, M16804, M16768, S73842, BC030554, BC017861. BC105589, S72525, S60175, S73838, S73835, S74779, Z22689, Z22687, Z22686, Z22685, Z22683, Z22682, Z22680, Z22679, Z22678, Z22677, Z22701, Z22676, X72500, Z22690, M89834, M89856, M89830, M89854, M89841 , M89828, AJ132840, AJ132839, AJ 132838, AJ132837, AJ132836, AJ132835, AJ132834, AJ132833, AJ 132832, AJ132831, AJ132790, AJ 132789, AJ 132788, AJ132787, AJ132786, AJ132785, AJ132784, AJ132783, AJ132783. Sequences of these are incorporated herein by reference.

[0078] Detection of TCR-γ gene rearrangement. The TCR-γ gene rearrangement may be detected by amplification. In one example, the amplification method is by PCR. The PCR method may include a primer pair where one primer of the primer pair hybridizes to a portion of Vγ region and the other primer of the primer pair hybπdizcs to a portion of Jγ region.

[0079] In one example, a consensus primer may be used for the members of the Vγ -1 subgroup due to high degree of sequence homology between its member of variable gene segments. In another example, a consensus primer may be used for Jγ 1 and Jγ 2 gene segments due to high degree of sequence homology between the two joining gene segments. In one specific example, the consensus primer Vγ -I subgroup is SEQ ID NO: 2 and the consensus primer for Jγ 1 / Jγ 2 subgroup is SEQ ID NO: 3. Sequences of SEQ ID NO: 2 and 3 are shown below:

[0080] 5 '-CAGGGTTGTGTTGGAATCAGG-S ' (SEQ ID NO: 2)

5 '-TGTTCCACTGCCAAAGAGTTTCTT-S ' (SEQ ID NO; 3)

[0081] Since many TCR-γ gene rearrangements involve Vγ -I subgroup of variable genes and Jγ 1 and Jγ 2 joining gene segments, SEQ ID NO: 2 and 3 may be used as a primer pair to amplify portions of TCR-γ gene comprising Vγ-J γ junctions in many cases.

[0082] Individual primers directed to each the variable regions subgroups Vγ -I , Vγ -II, Vγ -III, and Vγ -IV may be used along with primers directed to each of the joining gene segment subgroups Jγ 1 / Jγ 2; Jγ Pl / Jγ P2 and Jγ P to amplify portions of TCR-γ gene comprising the appropriate Vγ-Jγ junctions. In some cases, portions of TCR-γ rearranged gene comprising Vγ~~Jγ junctions may be amplified by multiplexed PCR. Alternatively, the multiplexed PCR reaction may use nested PCR primers. Exemplary sequences of the primers that may be used in the PCR reactions including multiplexed PCR and nested multiplexed PCR are shown in Table 2 below. The primers listed below are provided as examples. Other primers may be designed to hybridize to portions of variable and joining gene segments using the sequence of TCR-γ gene locus provided in SEQ ID NO: 1 as a guide. TABLE 2: Nucleic acid sequences of primers for PCR reaction

[0083] Amplifying portions of TCR-γ rearranged gene comprising V γ-J γ junctions is possible by nested PCR or hemi-nested PCR using combinations of primers listed above. For example, SEQ ID NO:4 and SEQ ID NO: 7 may be used as outer primer pair and SEQ ID NO: 10 and SEQ ID NO: 14 may used as an inner primer pair in a nested PCR reaction to amplify portions of TCR-γ rearranged gene where any of the gene segments of V γ -I subgroup is joined to J γ 1 or J γ 2 gene segments. Similarly, SEQ ID NO: 5 and SEQ ID NO: 8 may be used as outer primer pair and SEQ ID NO: 1 1 and SEQ ID NO: 15 may used as an inner primer pair in a nested PCR reaction to amplify portions of TCR-γ rearranged gene where any of the gene segment of V γ 9 is joined to J γ Pl or J γ P2.

[0084] Portions of TCR-γ gene comprising the Vγ-Jγ junctions may be detected using nucleic acid probes comprising a detectable label, In some instances, the probes may used to detect the amplicons generated during amplification reactions to amplify portions of TCR-γ gene comprising the Vγ-Jγ junctions. In other instances, the probes may be used in nucleic acid arrays to detect portions of TCR-γ gene. Exemplary sequences of probes are listed in Table 3. TABLE 3: Sequence of Probes

[0085] In one embodiment, the amplification reaction may include an internal control such as, for example, wild-type K-ras nucleic acid sequence. Full length and partial genomic sequences of human K-ras gene have been reported Exemplary sequences include but are not limited to NCBI GenBank accession numbers: EU332849, EF685662. EF685661 , EF471957, EF471953, CH471094, AC022509, NT__009714, NW_925328, NW 001838052, and NG 007524. These sequences are incorporated herein by reference. Exemplary sequence of human K-ras genomic DNA sequence is described by GenBank accession number NG 007524 Portion of human K-ras gene may be amplified using the pnmers SEQ ID NO: 33 and SEQ ID NO: 34. The oligonucleotide probe to detect a portion of human K- ras gene may be SEQ ID NO: 31. In another example, the oligonucleotide probe to detect a portion of human K-ras gene may be SEQ ID NO: 32 [0086J Detection of TCR-γ gene rearrangement in an acellular body fluid can be used for the diagnosis of lymphoproliferative disorder particularly when it is difficult to diagnose a lymphoproliferative disorder based only on clinical, histological and immunohistochemical criteria. Exemplary lymphoproliferative disorder includes but not limited to T-cell acute lymphoblastic leukemia (ALL), large granular lymphocytic leukemia, peripheral T-cell lymphoma, T-lymphoblastic lymphoma, T-cell prolymphocyte leukemia, γδ-hepatosplenic lymphoma, Sezary syndrome, mycosis fungoides (Gra et al. J MoI Diagn. 2007; 9: 249-257).

[0087] Sample

[0088] Sample may be of human or non-human origin. Sample may be obtained from an individual who is suspected of having a disease, or a genetic abnormality. In other cases, sample may be obtained from a healthy individual who is assumed of having no disease, or a genetic abnormality. In some examples, the sample may be obtained from an individual suspected of having lymphoproliferative disorder. In one example, the sample may be obtained from an individual suspected of having T-cell acute lymphoblastic leukemia (T-ALL). In some embodiments, the sample may be obtained from an individual diagnosed with lymphoma. In other embodiments, the sample may be obtained from an individual diagnosed with precursor B-leukemia.

[0089] An individual's plasma or serum may be used efficiently to detect TCR-γ nucleic acid and rearrangement of TCR-γ genes by the methods of the present invention. Detection of the rearrangement of TCR-γ genes in plasma of an individual has been found be at least as sensitive if not more so than detecting the same translocation from paired peripheral blood cells of the same individual.

[0090] Plasma or Serum Preparation Methods

[0091 ] Methods of plasma and serum preparation are well known in the art. Either "fresh" blood plasma or scrum, or frozen (stored) and subsequently thawed plasma or serum may be used. Frozen (stored) plasma or serum should optimally be maintained at storage conditions of -20 to -70 degrees centigrade until thawed and used. "Fresh" plasma or scrum should be refrigerated or maintained on ice until used, with nucleic acid (e.g., RNA, DNA or total nucleic acid) extraction being performed as soon as possible. Exemplary methods are described below.

[0092] Blood can be drawn by standard methods into a collection tube, preferably siliconized glass, either without anticoagulant for preparation of serum, or with EDTA, sodium citrate, heparin, or similar anticoagulants for preparation of plasma. Blood may be collected using commercially available kits and tubes such as BD Vacutainer® Push Button Blood Collection Set (BD, NJ, USA). Since heparin may interfere with RT-PCR, EDTA is the preferred anticoagulant for blood specimens in which PCR amplification is planned.

[0093] Nucleic Acid Extraction and Amplification

[0094] The nucleic acid (DNA or RNA) may be isolated from the sample according to any methods well known to those of skill in the art. If necessary the sample may be collected or concentrated by centrifugation and the like. In some embodiments, nucleic acid may be intact. Nucleic acid may be fragmented (e.g., digested with restriction endonucleases, or by sonication or by applying shearing force by methods known in the art).

[0095] Various methods of extraction are suitable for isolating the DNA or RNA. Suitable methods include phenol and chloroform extraction. See, Maniatis et al., Molecular Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press, page 16.54 (1989). Numerous commercial kits also yield suitable DNA and RNA including, but not limited to, QIAamp™ mini blood kit, Agencourt Genfind™, Roche Cobas® Roche MagNA Pure® or phenokchloroform extraction using Eppendorf Phase Lock Gels®, and the NucliSens extraction kit (Biomerieux, Marcy l'Etoile, France), Wizard Genomic DNA Kit (Cat.# A 1620, Promcga Corp. Madison, WI), Wizard SV Genomic DNA Kit (Cat.# A2360, Promega Corp. Madison, WI), the SV Total RNA Kit (Cat.# X3100, Promega Corp. Madison, WI), PolyATract System (Cat.# Z5420, Promega Corp. Madison, WI), or the PurYield RNA System (Cat.# Z3740, Promega Corp. Madison, WI). In other methods, mRNA may be extracted from patient blood/bone marrow samples using MagNA Pure LC mRNA HS kit and Mag NA Pure LC Instrument (Roche Diagnostics Corporation, Roche Applied Science, Indianapolis, IN). [0096] Extraction of DNA from acellular body fluids. DNA may be extracted from acellular body fluids, such as serum and plasma, using any appropriate method. Many suitable methods are known in the art including, for , In one such example, using the Qiamp Blood Kit (Qiagcn, Hildcn, Germany) according to the manufacturer's protocol for blood and body fluids, with the following modifications. First, heat about 7.5 to 12 ml of plasma at 99°C for 5 min on a heat block. Centrifuge the heated sample at 14,000 rpm for 30 min and collect the clear supernatant (about 1 ml) . Incubate the collected supernatant with Proteinase K (20 mg/ml; Boehringer Mannheim, Mannheim, Germany) and buffer AL (Qiagen) at a 1 : 10 ratio overnight at 55 ⁰ C. Finally, use a single column repeatedly to process a single sample, and quantify the purified DNA spectrophotometrically. Total DNA from plasma may be isolated using BioRobot® EZl automated nucleic acid purification workstation (QiaGen, CA, USA).

[0097] Extraction of RNA from acellular body fluids, RNA may be extracted from plasma or serum using silica particles, glass beads, or diatoms, as in the method or adaptations of Boom et al. J. Clin. Micro. 1990; 28: 495-503 and Cheung et al. J. Clin Micro. 1994; 32: 2593-2597. As an alternative method, RNA may be extracted from plasma or serum using the Acid Guanidinium Thiocyanate-Phenol-chloroform extraction method described by Chomczynski, P. and Sacchi, N., Anal. Biochem. 1987; 162: 156-159. Trace amounts of contaminating DNA may be removed from RNA prior to proceeding to further RNA analysis. This may be accomplished using DNase, for example by the method as described by Rashtchian, A. PCR Methods Applic. 1994; 4: S83-S91. These methods are incorporated herein by reference.

[0098] Detection of target nucleic acid

[0099] Detection of nucleic acid by size. Methods for detecting the presence or amount of polynucleotides are well known in the art and any of them can be used in the methods described herein so long as they are capable of separating individual polynucleotides by the difference in size of the nucleic acid. The separation technique used should permit resolution of nucleic acid as long as they differ from one another by at least one nucleotide. The separation can be performed under denaturing or under non-denaturing or native conditions-- i.e., separation can be performed on single- or double-stranded nucleic acids. Useful methods for the separation and analysis of the amplified products include, but are not limited to, electrophoresis (e.g., agarose gel electrophoresis, capillary electrophoresis (CE)), chromatography (HPLC), and mass spectrometry.

[00100] CE provides exceptional separation of the polynucleotides in the range of at least 10-1 ,000 base pairs with a resolution of a single base pair. CE can be performed by methods well known in the art, for example, as disclosed in U.S. Pat. Nos. 6,217,731 ; 6,001,230; and 5,963,456, which are incorporated herein by reference. High-throughput CE apparatuses arc available commercially, for example, the HTS9610 High throughput analysis system and SCE 9610 fully automated 96-capillary electrophoresis genetic analysis system from Spectrumedix Corporation (State College, Pa.); P/ACE 5000 series and CEQ series from Beckman Instruments Inc (Fullerton, Calif.); and ABI PRISM 3100 genetic analyzer (Applied Biosystems, Foster City, Calif). Near the end of the CE column, in these devices the amplified DNA fragments pass a fluorescent detector which measures signals of fluorescent labels. These apparatuses provide automated high throughput for the detection of fluorescence-labeled PCR products.

[00101] HPLC may used to analyze nucleic acids such as single and double stranded DNA based on their size. Methods for separation of single and double stranded DNA by HPLC are known in the art. Nucleic acid may be purified by HPLC using either anion exchange or reverse phase columns. Typically reverse phase HPLC columns include C8, C 14, C 18 columns (Waters, MA, USA, Agilent. CA, USA), or poly (styrene-divinylbenzene) columns such as PRP-I and PRP-3 (Hamilton Company, NV, USA) may be used. When reverse phase columns are used, the nucleic acid bound to the column may be eluted with a gradient of increasing concentration of hydrophobic solvent such as acctonitrilc. Several variations of mobile phases are possible to elute the bound nucleic acid from reverse phase columns such as: Buffer A) 50 mM Sodium Hydroxide pH 12.7, Buffer B) 1 :1 50 mM Sodium Hydroxide : Acetonitrile (Germann et al. Anal Biochem. 1987; 156: 399); Buffer A) 10 mM Potassium Phosphate pH 7, Buffer B) 7: 1 :2 V: V: V Acetonitrile : Methanol : 10 mM Potassium Phosphate pH 7 (Arghavani et al. Anal Biochem. 1995; 231 : 201 ). An exemplary method is described by Glfar et al. (Waters Application note 2007; 720002376en). The method of Glfar et al. separates 50-600 bases of double stranded DNA using a solvent system with two mobile phases. Mobile phase A comprise 0. IM triethylammonium acetate and Mobile phase B comprise 20% acetonitrile in 0.1 M triethylammonium acetate. A C18 column (2.1 X 50 mm, 1.7 μM) column with a pore size of 300 A ^Q was used for this purpose. The gradient used for separation is 57.5-84.5% B in 20 minutes. The separation of DNA was monitored at 260 ran. This method is incorporated herein by reference. Other HPLC based methods for analysis of nucleic acids (both DNA and RNA) are described by Wang et al. (J MoI Diagn. 2007; 9(4): 441-451 ); Chen ct al. (Chem Res Toxicol. 2008); Kim et al. (RNA. 2007; 13(2): 289-294), Reddy et al. American Lab. 1995; 15: 15. All of these methods are incorporated herein by reference.

[00102] Exemplary anion exchange HPLC column for analysis of nucleic acid include PRP-X600 (Hamilton Company, NV, USA). Exemplary purification condition include Conditions: Mobile phase A) 20 mM TRIS, 1 mM EDTA pH 9.0; B) IN Sodium Chloride in 20 mM TRIS, 1 mM EDTA; linear gradient: 60-67.5% B (0-15 min), 67.5-75% B (15-45 min) at 1.0 mL/min, Ambient. UV at 260 nm. Another exemplary purification condition include mobile phase: A) 85: 15 100 mM TRIS pH 8.0 :Acetonitrilc; B) 85: 15 100 mM TRIS pH 4.0,2.5 M Lithium Chloride : Acetonitrile; linear gradient 0-100% B for 10 min at 2.0 mL/min; 85°C, UV at 260 nm.

[00103] Nucleic acid may be also be analyzed and detected by size using agarose gel electrophoresis. Methods of performing agarose gel electrophoresis are well known in the art. See Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Ed.) (1989), Cold Spring Harbor Press, N. Y.

[00104] DNA sequencing. Detection of nucleic acid is possible by DNA sequencing. Sequencing may be carried out by the dideoxy chain termination method of Sanger et al.(Proc. Natl. Acad. Sci. USA 1977; 74: 5463-5467) with modifications by Zimmermann et al. Nucleic Acids Res. 1990; 18: 1067. Sequencing by dideoxy chain termination method can be performed using Thermo Sequenase (Amersham Pharmacia, Piscataway, NJ), Sequenase reagents from US Biochemicals or Sequatherm sequencing kit (Epicenter Technologies, Madison, Wis.). Sequencing may also be carried out by the "RR dRhodamine Terminator Cycle Sequencing Kit" from PE Applied Biosystems (product no. 403044, Weitcrstadt, Germany), Taq DyeDeoxy™ Terminator Cycle Sequencing kit and method (Perkin- Elmer/ Applied Biosystems) in two directions using an Applied Biosystems Model 373A DNΛ or in the presence of dye terminators CEQ ^{1 M} Dye Terminator Cycle Sequencing Kit, (Beckman 608000).

[00105] Alternatively, sequencing can be performed by a method known as Pyrosequencing (Pyrosequencing, Westborough, Mass.). Detailed protocols for Pyrosequencing can be found in: Λlderbom et al. Genome Res. 2000; 10: 1249-1265.

[00106] Detection of target nucleic acid by probes. Probes are capable of hybridizing to at least a portion of the target nucleic acid for example, a portion of TCR-γ nucleic acid, a portion of K-ras nucleic acid, or to any other target sequence of interest. In some cases, the probe can hybridize to a portion of Vγ region and to a portion of Jγ region.

[00107] Probes can be about 10 bases, about 20 bases, about 30 bases, about 40 bases, about 50 bases, about 60 bases, about 75 bases, about 100 bases, about 150 bases, about 200 bases.

[00108] However, probes can be longer. Longer probes can be from few hundred bases to few million bases. In one embodiment, the nucleic acid probes are derived from one, several or all of the human genomic nucleic acid segments provided in a compendium of bacterial artificial chromosomes (BΛCs) compiled by The BΛC Resource Consortium, (see McPherson et al., Nature 409:934-41, 2001).

[001 09] Probes consist of a detectable label or a plurality of detectable labels. The detectable label associated with the probe can generate a detectable signal directly. Alternatively, the detectable label associated with the probe can be detected indirectly using a reagent, in which the reagent includes a detectable label, and binds to the label associated with the probe. In one example, the reagent includes a detectable label is a labeled antibody. In another example, the reagent including a detectable label is a primary antibody/secondary antibody pair, in which the detectable label may be in the primary antibody, or in the secondary antibody or in both.

[00110] Probes can be TaqMan® probes, molecular beacons, and Scorpions (e.g., Scorpion™ probes). These types of probes are based on the principle of fluorescence quenching and involve a donor fluorophore and a quenching moiety. The term '"fluorophore" as used herein refers to a molecule that absorbs light at a particular wavelength (excitation frequency) and subsequently emits light of a longer wavelength (emission frequency). The term "donor fluorophoie" as used herein means a fluorophore that, when in close proximity to a quencher moiety, donates or transfers emission energy to the quencher. As a result of donating energy to the quencher moiety, the donor fluorophore will itself emit less light at a particular emission frequency that it would have in the absence of a closely positioned quencher moiety.

[001 1 1] The term "quencher moiety" as used herein means a molecule that, m close proximity to a donor fluorophore, takes up emission energy generated by the donor and either dissipates the energy as heat or emits light of a longer wavelength than the emission wavelength of the donor In the latter case, the quencher is considered to be an acceptor fluorophore. The quenching moiety can act via proximal (i.e., collisional) quenching or by Fόrster or fluorescence resonance energy transfer ("FRET") Quenching by FRET is generally used in TaqMan® probes while proximal quenching is used in molecular beacon and Scorpion™ type probes. Suitable quenchers are selected based on the fluorescence spectrum of the particular fluorophore. Useful quenchers include, for example, the Black Hole™ quenchers BHQ- I , BHQ-2, and BHQ-3 (Biosearch Technologies, Inc.), and the ATTO-series of quenchers (ATTO 540Q, ATTO 580Q, and ATTO 612Q; Atto-Tec GmbH).

[00112] With Scorpion pnmers, sequence-specific priming and PCR product detection is achieved using a single molecule The Scorpion primer maintains a stem-loop configuration in the unhybπdi7ed state The fluorophore is attached to the 5' end and is quenched by a moiety coupled to the 3' end, although in suitable embodiments, this arrangement may be switched The 3' portion of the stem also contains sequence that is complementary to the extension product of the primer. This sequence is linked to the 5' end of a specific primer via a non-amphfiable monomer. After extension of the primer moiety, the specific probe sequence is able to bind to its complement within the extended amplicon thus opening up the hairpin loop. This prevents the fluorescence from being quenched and a signal is observed A specific target is amplified by the reverse pπmer and the primer portion of the Scorpion primer, resulting m an extension product. A fluorescent signal is generated due to the separation of the fluorophore from the quencher resulting from the binding of the probe element of the Scorpion pπmer to the extension product [001 13] TaqMan® probes (Held, et al , Genome Res 1996, 6 986-994) use the fluorogenic 5' exonuclease activity of Taq polymerase to measure the amount of target sequences in cDNA samples TaqMan® probes are oligonucleotides that contain a donor fluorophore usually at or near the 5' base, and a quenching moiety typically at or near the 3' base The quencher moiety may be a dye such as TAMRA or may be a non-fluorescent molecule such as 4-(4 -dimethylammophenylazo) benzoic acid (DABCYL) See Tyagi et al Nature Biotech 1998, 16 49-53 When irradiated, the excited fluorescent donor transfers energy to the nearby quenching moiety by FRET rather than fluorescing Thus, the close proximity of the donor and quencher prevents emission of donor fluorescence while the probe is intact

[001 14J TaqMan® probes are designed to anneal to an internal region of a PCR product When the polymerase (e g , reverse transcriptase) replicates a template on which a TaqMan® probe is bound, its 5' exonuclease activity cleaves the probe This ends the activity of the quencher (no FRET) and the donor fluorophore starts to emit fluorescence which increases m each cycle proportional to the rate of probe cleavage Accumulation ol PCR product is detected by monitoring the increase in fluorescence of the reporter dye (note that primers are not labeled) If the quencher is an acceptor fluorophore. then accumulation of PCR product can be detected by monitoring the decrease in fluorescence of the acceptoi fluorophore

[001 15] Detection by Hybridization Nucleic acid such as TCR-γ can be detected by hybridization to a nucleic probe The methods of the present invention can incorporate all known methods and means and variations thereof for carrying out DNA hybridization, see, e g , Sambrook, et al , 1989, Molecular Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plamview, NY

[001 16] 1 he nucleic acid of interest may form a complex on a solid support prior to being detected The complex may comprise a capture probe anchored to a solid support, the nucleic acid of interest hybridized to the capture probe, and a detectably labeled probe hybridized to the nucleic acid of interest In some cases, the solid support may comprise a first member of a binding pair and the capture probe may comprise a second member of the binding pair The binding of the first member of the binding pair to the second member of the binding pair may anchor the capture probe to the solid support Examples of solid support include but are not limited to beads, mi crop articles, microarray plates, microwells Examples of binding pair include but are not limited to biotin/streptavidm, hgand-receptor, hormone-receptor, and antigen-antibody

[00 I I ⁷ ] I CR-γ gene rearrangement can be detected by performing an array-based comparative genomic hybridization (CGH) to detect the rearrangement of the TCR-γ genes in a sample, or to diagnose a genetic abnormality in an individual The resolution of array- based CGH is primarily dependent upon the number, size and map positions of the nucleic acid elements within the array, which are capable of spanning the entire genome Bacteπal artificial chromosomes, or BACs, which can each accommodate on average about 150 kilobases (kb) of cloned genomic DNA, may be used in the production of the array Alternatively, smaller oligonucleotide probes capable of hybridizing to the rearranged TCR- γ genes may be utilized

[001 18] The hybπdized complexes can also be detected using flow cytometry Flow cytometry is a technique well-known in the art Flow cytometers hydrodynamically focus a liquid suspension of particles (c g , cells or synthetic microparticles or beads) into an essentially single-file stream ot particles such that each particle can be analyzed individually Flow cytometers are capable of measuring forward and side light scattcπng which correlates with the size of the particle Thus, particles of diffeπng sizes may be used in invention methods simultaneously to detect distinct nucleic acid segments In addition fluorescence at one or more wavelengths can be measured simultaneously Consequently, particles can be sorted by size and the fluorescence of one or more fluorescent labels probes can be analyzed for each particle Exemplary flow cytometers include the Becton-Dickenson Immunocytometry Systems FACSCAN Equivalent flow cytometers can also be used in the invention methods

[001 19] Detection is possible by Southern blotting and hybridization with a labeled probe The techniques involved in Southern blotting are well known to those of skill in the art and may be found in many standard books on molecular protocols See, Sambrook et al , Molecular Cloning A Laboratory Manual (2nd Ed ) (1989), Cold Spring Harbor Press, N Y [00120] Detectable label

[00121] The term "detectable label" as used herein refers to a molecule or a compound or a group of molecules or a group of compounds associated with a probe and is used to identify the probe hybridized to a genomic nucleic acid or reference nucleic acid.

[00122] Detectable labels include but are not limited to fluorophores, isotopes (e.g. 32P, 33P,35S, 3H, 14C, 1251, 1311), electron-dense reagents (e.g., gold, silver), nanoparticlcs, enzymes commonly used in an ELISA (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminiscent compound, colorimetric labels (e.g., colloidal gold), magnetic labels (e.g., DynabeadsTM), biotin, digoxigenin, haptens, proteins for which antisera or monoclonal antibodies are available, ligands, hormones, oligonucleotides capable of forming a complex with the corresponding oligonucleotide complement.

[00123] In some embodiments, the detectable label is a fluorophore. Suitable fluorescent moieties include but are not limited to the following fluorophores working individually or in combination:

4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; Alexa Fluors: Alexa Fluor ^® 350, Alexa Fluor ^® 488, Alexa Fluor ^® 546, Alexa Fluor ^® 555, Alexa Fluor ^® 568, Alexa Fluor ^® 594, Alexa Fluor ^® 647 (Molecular Probes); 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS); 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4-anilino-l- naphthyl)maleimide: anthranilamide; Black Hole Quencher™ (BHQ ^rM ) dyes (biosearch Technologies); BODIPY dyes: BODIPY ^® R-6G, BOPIPY ^® 530/550, BODIPY ^® FL; Brilliant Yellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluariπ (Coumarin 151 ); Cy2 ^® , Cy3 ^® , Cy3.5 ^® , Cy5 ^£ , Cy5.5*; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5"-dibromopyrogallol- sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4- methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'- disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5- [dimethylamino]naphthalene-l-sulfonyl chloride (DNS, dansyl chloride); 4-(4'- dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'- isothiocyanate (DABITC); Eclipse™ (Epoch Biosciences Inc.): eosin and derivatives: eosin, eosin isothiocyanate; erythrosin and derivatives: erythrosin B, erythrosin isothiocyanate; ethidium; fluorescein and derivatives: 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2- yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-ό-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), hexachloro-6-carboxyfluorescein (HEX), QFITC (XRITC), tetrachlorofluorescein (TET); fluorescaminc; IR 144; IR1446; lanthamidc phosphors; Malachite Green isothiocyanate; 4-m ethyl umb el liferone; ortho cresolphthalein; nitrotyrosine: pararosaniline; Phenol Red; B-phycoerythrin, R-phycoerythrin; allophycocyanin; o-phthaldialdehyde; Oregon Green*; propidium iodide; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1 -pyrene butyrate; QSY* 7; QSY* 9; QSY ^® 21 ; QSY ^β 35 (Molecular Probes); Reactive Red 4 (Cibacron ^® Brilliant Red 3B-A); rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamine X isothiocyanate, riboflavin, rosolic acid, sulforhodamine B, sulforhodamine 101 , sulfonyl chloride derivative of sulforhodamine 101 (TEXAS RED®); terbium chelate derivatives; N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC).

[00124] Kks

[00125] The present inventions also contemplate diagnostic systems in kit form. A diagnostic system of the present inventions may include a kit which contains, in an amount sufficient for at least one assay, any of the hybridization assay probes, amplification primers, for detecting TCR-γ nucleic acid and TCR-γ gene rearrangement in a packaging material. Typically, the kits will also include instructions recorded in a tangible form (e.g., contained on paper or an electronic medium) for using the packaged probes, primers, and/or antibodies in a detection assay for determining the presence or amount of TCR-γ nucleic acid and TCR- y gene rearrangement in a test sample.

[00126] The various components of the diagnostic systems may be provided in a variety of forms. For example, the required enzymes, the nucleotide triphosphates, the probes, primers, and/or antibodies may be provided as a lyophilized reagent. These lyophilized reagents may be pre-mixed before lyophilization so that when reconstituted they form a complete mixture with the proper ratio of each of the components ready for use in the assay. In addition, the diagnostic systems of the present inventions may contain a reconstitution reagent for reconstituting the lyophilized reagents of the kit. In preferred kits for amplifying target nucleic acid derived from an individual, the enzymes, nucleotide triphosphates and required cofactors for the enzymes are provided as a single lyophilized reagent that, when reconstituted, forms a proper reagent for use in the present amplification methods.

[00127J In one embodiment, the kit may comprise at least three lyophilized oligonucleotides: a primer pair to amplify a portion of TCR-γ nucleic acid and a portion of nucleic acid comprising TCR-γ gene rearrangement and a detectably labeled probe capable of hybridizing to the amplicon generated. In some preferred kits, at least two lyophilized oligonucleotides: the primer pair for amplification of at least a portion of nucleic acid comprising TCR-γ gene rearrangement may have sequences of SEQ ID NO: 2, 3, or complements and fragments thereof respectively. In some embodiments, the kit may comprise primers and probes for internal control. In one embodiment, the kit may comprise primers and probes for amplification and detection of human K-ras gene. In one embodiment, the kit may comprise oligonucleotide probes SEQ ID NO: 31 and SEQ ID NO: 32. In another embodiment, the kit may comprise primers for amplifying a portion of K-ras gene: SEQ ID NO: 33 and SEQ ID NO: 34.

[00128] Some preferred kits may further comprise to a solid support for anchoring the nucleic acid of interest on the solid support. The target nucleic acid may be anchored to the solid support directly or indirectly through a capture probe anchored to the solid support and capable of hybridizing to the nucleic acid of interest. Examples of such solid support include but arc not limited to beads, microparticles (for example, gold and other nano particles), microarray, microwells, multiwell plates. The solid surfaces may comprise a first member of a binding pair and the capture probe or the target nucleic acid may comprise a second member of the binding pair. Binding of the binding pair members will anchor the capture probe or the target nucleic acid to the solid surface. Examples of such binding pairs include but are not limited to biotin-'streptavidin, hormone/receptor, ligand/receptor, antigen/antibody. [00129] The versatility of the inv eπtion is illustrated by the following Examples which illustrate preferred embodiments of the invention and are not limiting of the claims or specification in any way

EXAMPLE 1

[00130] Sample Collection

[00131] Blood was collected in ED 1 A-containmg tubes (Becton Dickinson, NJ) from 215 individuals suspected with lymphoid malignancies (based on tissue biopsies and typical lymphoid infiltrate diagnosis) and 195 individuals without lymphoid malignancies Plasma was separated from blood cells by differential centπfugation at 1000 X g for 1 5 mm Respective blood cells were separated from RBC by differential centπfugation using Puregene® RBC lysis solution (Gentra Systems, MN, USA) The cell pellet was washed with phosphate-buffered salme Both plasma and cell samples were cryopreserved at -8O ⁰ C for future use

[00132] Total DNA from plasma and cell samples were isolated using BioRobot® EZl automated nucleic acid purification workstation (QiaGen, CA, USA)

EXAMPLE 2

[00133] PCR amplification of isolated DNA

[00134] PCR amplification was performed in triplicate for K-ras gene, IgH gene, IgH/Bcl- 1 translocation, TCR-γ, and IgH ^/ Bcl-2 translocation using the pnmers listed in Table 4 PCR was performed using ABI 7900 detection system and the PCR conditions discussed below For detection of TCR-γ gene rearrangement, PCR pnmers SEQ ID NO 2 and 3 were used In each experiment, amplification of the K-ras gene serv ed as an mterndl positive control and sterile water served as negative control and used as base line TABLE 4. Sequences of primers and probes used in PCR analysis

Internal reference gene Primer

K-ras-F probe 5'-FAM-ATGACTGAATATAAAC ITGT-3'

(SEQ ID NO: 31)

K-ras-R probe 5'-F AM-TGGTAGTTGGAGCTGGTGGCGTA-T AMRA-3 ¹

(SEQ ID NO: 32)

K-ras-F 5 '-GCCTGCTG AAAATG ACTG AAT-3 '

(SEQ ID NO: 33)

K-ras-R 5'-GGTCCTGCACCAGTAAT ATGC-3'

(SEQ TD NO: 34)

IgII chain gene

J _H -FAM 5'-FAM-ACCTGAGGAGACGGTGACC-3' (SEQ ID NO: 35)

FR2a 5'-TGGRTCCGMCAGGCYCNGG-S' (SEQ ID NO: 36) FR3a 5'-TGTCGACACGGCYSTGTΛTTΛCTG-3' (SEQ ID NO: 37)

V,,-FR3a-FAM 5'-FAM-ACACGGCCGTGTATTACTG-3' (SEQ ID NO: 38) J _H -CDR3 5'-GTGACCAGGGTNCCTTGGCCCCAG-S' (SEQ ID NO: 39)

TCR-γ chain gene

TcellV-F-FAM 5'-FAM-CAGGGTTGTGTTGGAATCAGG-S' (SEQ ID NO: 2)

TcellJ-R S'-TGTTCCACTGCCA.ΛAGAGTTTCTT-S' (SEQ ID NO: 3)

BCL l gene

BCL-I MTC-F 5'-TGGATAAAGGCGAGGAGCATAA-S' (SEQ ID NO: 40)

BCL-I MTC-F probe 5'-FAM-ACTGCATATTCGGTTAGACTGTGATTAGCTTT- TAMRA-3'

(SEQ TD NO: 41 ) J _H -BCL-I -R 5'-ACCTGΛGGAGACGGTGACC-3' (SEQ ID NO: 42) BCL-2 gene

BCL-2 MBR-F 5'-TTAGAGAGTTGCnTACGTGGCC-S ¹

(SEQ ID NO; 43)

BCL-2 MCR-F 5'-CCTGGCTTCCTTCCCTCTGT-S'

(SEQ ID NO; 44) BCL-2 MBR probe 5'-FAM-CAGGAGGGCTCTGGGTGGGTCTGT-TAMRA-S'

(SEQ ID NO: 45) BCL-2 MCR probe 5'-FAM-TGTCCTTCCTTTCCACTCCTCCCCAGA-TAMRA-S'

(SEQ ID NO: 46) JH-BCL-2-R 5'-ACCTGAGGAGACGGTGACC-S'

(SEQ ID NO: 30)

[00135] PCR conditions

[00136] For IgH gene: 94 ⁰ C for 8 min, 52 ⁰ C for 20 sec, and 72 ° C for 5 min. Cycle repeated for 35 times. For TCR-γ gene: 94°C for 8 min, 60 ⁰ C for 90 sec, 72 ⁰ C for 10 min. Cycle repeated for 35 times. For IgH/Bcl-1 translocation: 95 ⁰ C for 10 min (1 ^SI cycle); 95 ⁰ C for 15 sec, 60 ⁰ C for 1 min. Cycle repeated 44 times. For IgH/Bcl-2 translocation: 95 ⁰ C for 10 min (1 ^st cycle); 95 ⁰ C for 15 sec, 60 ⁰ C for 1.5 min. Cycle repeated 44 times. For K-ras, the PCR condition was same as the individual genes indicated above.

EXAMPLE 3

[00137] Analysis of the PCR amplified fragments.

[00138] PCR amplified fragments were analyzed by capillary electrophoresis using ABI PRISMCB ⁾ 3100 genetic analyzer (Applied Biosystems, CA, USA). The results of the separation by capillary electrophoresis of the amplified products from plasma and cell samples are shown in Fig. 2. The sizes of the products are indicated in the top of the spectrum (ranging from 30 bases to over 540 bases). The relative number/amount of each product size is indicated on the y-axis. The size of the K-ras amplified product was 108-bp.

[00139] Clonal rearrangement of the IgH gene resulted in predominant products with a size range of 220-310 bp for FR2a ^/ J _H primer pair, 70-150 bp for FR3/Jh primer pair and 50- 140 bp for FR3a/CDR3 primer pair for monoclonal population in both cell and plasma samples (Fig. 2A). For polyclonal IgH cell and plasma samples, multiple peaks were observed between the product sizes 220-310 bp indicating the presence of multiple rearranged IgH genes.

[00140] Clonal rearrangement of the TCR-γ gene resulted in predominant products with a size range of 140-180 bp using primer pairs SEQ ID NO: 2 and 3 with one major peak and one minor peak were observed for monoclonal population in both cell and plasma samples (Fig. 2B). For polyclonal TCR-γ cell and plasma samples, multiple peaks were observed between the 140-180 bp product sizes indicating the presence of multiple rearranged TCR-γ genes.

EXAMPLE 4

[00141] Sensitivity of detection of nucleic acids comprising IgH, TCR-γ, IgH/Bcl-1 translocation and IgH/Bcl-2 translocation in acellular body fluid

[00142] To analyze the sensitivity of detection of nucleic acids in plasma, PCR amplification followed by capillary electrophoresis was performed on nucleic acids isolated from paired plasma and peripheral blood cells (PB) and tested for IgH gene rearrangement (B-cell clonality) and TCR-γ gene rearrangement (T-cell clonality), IgH/Bcl-1 translocation and IgH/Bcl-2 translocation. Clonal rearrangement of the IgH gene resulted in predominant products with a size range of 220-310 bp for FR2a/Jn primer pair, 70-150 bp for FR3/Jh primer pair and 50-140 bp for FR3a/CDR3 primer pair. Clonal rearrangement of the TCR-γ gene resulted in predominant products with a size range of 140-180 bp using primer pairs SEQ ID NO: 2 and 3 and shown in Fig. 2. Results from paired plasma and peripheral blood cells were compared. The results arc shown in Table 5 below.

TABLE 5. Correlation between matched plasma and peπpheral blood (PB) cell samples for 4 lymphoid malignancy-specific gene rearrangements

* All comparisons used Fisher's exact test.

The numbers of healthy subjects included as the control group in IgH, TCR-y, BCL-I, and

BCL-2 studies are 54, 35, 52 and 54, respectively.

[00143] A 100% concordance was observed for B-cell clonality between results from PB cells and plasma. 17 of 57 cases were identified as monoclonal and rest were identified as polyclonal population.

[00144] Plasma and PB cell analysis yielded 100% concordance for determination of T- cell clonality in 57 patients. 17 of the 57 patients (30%) were monoclonal population.

[00145] Unless otherwise defined, 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 nucleotide sequences provided herein arc presented in the 5' to 3 ' direction.

[00146] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc, shall be read expansively and without limitation Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that vaπous modifications are possible within the scope of the invention claimed.

[00147] Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

[00148] The invention has been described broadly and generically herein. Each of the narrower species and subgeneπc groupings falling within the generic disclosure also form part of the invention. 1 his includes the generic descπption of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[00149] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recogni/e that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group

[00150] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control

[00151] Other embodiments are set forth within the following claims