Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-06-21
2004-04-13
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091210, C435S091500, C536S023100, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06720144
ABSTRACT:
INTRODUCTION
This invention relates to the field of detecting a genetic marker of T-cell malignancies. The invention provides compositions and methods useful for detecting clonal T-cell receptor gene arrangements. By using PCR and temporal temperature gradient gel electrophoresis (TTGE), the clonality of T-cell populations in a sample can be determined.
BACKGROUND OF THE INVENTION
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 present invention.
T lymphocytes are important players in the immune response of mammalian organisms. Each T-lymphocyte has on its surface a molecule, known as the T-cell receptor (“TCR”) that exhibits a specificity of binding to a target antigen that is similar to the specificity of binding exhibited by antibodies, and TCR is encoded by genes having an organization similar to that of antibody genes.
During normal T-cell development, each TCR gene (&agr;, &bgr;, &ggr; and &dgr;) can rearrange, leading to highly diverse TCR proteins. Among the four TCR gene types, the TCR-&ggr; genes are the first to rearrange. During the rearrangement of each TCR gene, one of its different variable (V) regions combines with one of its different joining (J) regions. In the TCR-&ggr; gene, a few nucleotides (N region) are also inserted randomly into the VJ regions by terminal nucleotidyl tansferase (TdT), resulting in an increased sequence diversity in the TCR-&ggr; gene as compared to the other TCR genes. It is believed that each mature T-cell possesses an individual sequence of rearranged TCR genes.
T-cell malignancies can present to the physician with strikingly similar clinical patterns to simple reactive, or inflanmatory, diseases. T-cell malignancies, however, can be differentiated from reactive diseases by the presence in malignancy of an overgrowth of a single, clonal, T-cell population. Therefore, analysis of TCR gene rearrangement, and particularly the TCR-&ggr; gene, is of practical value in determining the clonality T-cell populations for the diagnosis and prognosis of T-cell malignancies (Flug et al., Proc Nat1 Acad Sci USA. 82: 3460-3464, 1985; Yanagi et al., Nature 308: 145-149, 1984; Waldmann et al., N Engl J Med 313: 776-783, 1985; Raulet, Annu Rev Immunol. 7: 175-207, 1989; Theodorou et al., Blood 86: 305-310,1995).
To date, numerous techniques have been used to analyze TCR gene rearrangement. Southern blotting hybridization was the first widely used technique (Spagnolo et al., Pathol. 26: 268-275, 1994). Because Southern blotting hybridization can be time-consuming and labor-intensive, and requires the use of large amounts of high molecular weight DNA and radioactivity, polymerase chain reaction (PCR)-based techniques have become more popular in research and clinical laboratories. These techniques can also permit the use of DNA extracted from formalin fixed parafin-embedded specimens and are more rapid than hybridization approaches (Theodorou et al., Blood 86: 305-310,1995; Murphy et al., J Cutan Pathol. 27: 228-234, 2000; Anderson et al, J. Cutan Pathol. 26: 176-182, 1999; Wood et a., J Invest Dermatol. 103: 34-41,1994; Menke etal., Electrophoresis 16: 733-738, 1995; Bourguin et al., Proc Natl Acad Sci USA. 87: 8536-8540, 1990).
PCR amplification of one or more TCR genes from non-malignant peripheral blood or lymph node tissue samples generates a mixture of multiple DNA molecules differing in size and/or base pair composition. Because the combinatorial and junctional diversity in TCR gene rearrangement is limited, the amplified products may be of similar length. Thus, the limited diversity may result in false clonal bands when analyzed by a standard gel electrophoresis methods that separate DNA molecules based solely on size (Theodorou et al., Blood 86: 305-310,1995; Menke et al., Electrophoresis 16: 733-738, 1995), and can occur when using either Southern blotting hybridization (which is based on agarose gel electrophoresis) or PCR followed by standard polyacrylamide gel electrophoresis (PAGE). Furthermore, due to the poor resolution of standard gel electrophoresis methods, DNA bands of low frequency clones may be lost in the polyclonal background “smear,” leading to difficult in interpreting the results of such an analysis.
Recently, electrophoresis techniques that resolve DNA molecules based on size and base pair composition have been explored, such as polymorphism (SSCP) (Murphy et al., J Cutan Pathol. 27: 228-234, 2000), denaturing gradient gel electrophoresis (DGGE) (Theodorou et al., Blood 86: 305-310, 1995; Anderson et al., J. Cutan Pathol. 26: 176-182, 1999; Wood et al., J Invest Dermnatol. 103: 34-41, 1994) and temperature gradient gel electrophoresis (TGGE) (Menke et al., Electrophoresis 16: 733-738, 1995). To maximize the resolution of SSCP, however, more than one electrophoretic condition is often needed (Orita et al., Genomics 5: 874-879, 1989). Moreover, although DGGE has been gaining popularity to determine the clonality of T-cell populations (Theodorou et al., Blood 86: 305-310,1995; Anderson et al., J. Cutan Pathol. 26: 176-182, 1999; Wood et al., J Invest Dermatol. 103:34-41, 1994), the difficulty in preparing denaturing gradient polyacrylamide gel limits the routine usage of this technique in clinical laboratories. Similarly, the routine use of TGGE in clinical laboratories is also limited because of its reliance on an instrument that is both expensive and difficult to maintain. (Menke et al. Electrophoresis. 16:733-738, 1995; Alkan et al. Arch Pathol Lab Med. 125:202-207, 2001).
Thus, there remains in the art a need for methods and compositions that can reproducibly and economically resolve clonal T-cell receptor populations in patient samples.
SUMMARY OF THE INVENTION
In the present invention provides methods and compositions to determine the clonality of T-cell receptor populations present in a sample. Temporal temperature gradient gel electrophoresis (TTGE), developed by Yoshino et al. (Nucleic Acids Res. 19:3153, 1991) and Chen et al. (Clin Chem. 45:1162-1167, 1999), can be used to detect the presence or absence of a clonal TCR gene rearrangement. In the instant methods, target DNA is electrophorcsed in a denaturing gel, and the temperature of the gel is increased gradually and uniformly across a range in which differences in the base composition of the target DNA are resolved. Because the gel itself is not a gradient gel, and because the temperature gradient is temporal rather than spatial across the gel, the instant methods can be performed in an economical fashion. In addition, the resulting DNA band patterns can be easily interpreted and quality controlled in a clinical laboratory setting.
In accordance with the present invention, TTGE can detect clonal TCR (e.g., TCR-&ggr;) gene arrangements present in a sample at concentrations as low as one malignant T-cell among 100 normal cells. Additionally, DNA can be extracted from stored samples (e.g., samples stored at 4° C. for up to 7 days and at room temperature for up to 4 days) and used for PCR amplification to generate gene amplicons for TCR gene rearrangement analysis by TTGE without significant variability of the band pattern and signal intensity. Thus, the invention provides simple, accurate and sensitive techniques to diagnose and monitor patients with T-cell malignancies.
In a first aspect, the instant invention relates to methods for determining the clonality of a T-cell receptor (TCR) rearrangement in a sample comprising the steps of extracting nucleic acids from the sample; amplifying the nucleic acids, preferably by polymerase chain reaction with one or more TCR specific primers to provide one or more TCR DNA fragments; and analyzing amplified TCR DNA fragments using an electrophoretic gel by temporal temperature gradient gel electrophoreis (TTGE). The present of one or more discrete bands in the electrophoretic gel indicates the presence of a clonal TCR rearrangement.
In certain embodiments, the electrophoretic profile of a
Samoszuk Michael
Zhu Dan
Foley & Lardner
Fredman Jeffrey
Quest Diagnostics
Warburg Richard J.
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