Detection of Clonal T-Cell Receptor-gamma Gene Rearrangement by PCR/Temporal Temperature Gradient Gel Electrophoresis

Transcription

1 Hematopathology / DETECTION OF CLONAL T-CELL RECEPTOR-GAMMA GENE REARRANGEMENT Detection of Clonal T-Cell Receptor-gamma Gene Rearrangement by PCR/Temporal Temperature Gradient Gel Electrophoresis Dan Zhu, MD, PhD, 1 Marshall E. Kadin, MD, 2 and Michael Samoszuk, MD 1 Key Words: T-cell lymphoma; Polymerase chain reaction; PCR; Temporal temperature gradient gel electrophoresis; TTGE Abstract Limited combinatorial and junctional diversity in TCR-gamma gene rearrangement can result in amplification products that are difficult to interpret when analyzed by conventional gel electrophoresis methods that separate DNA based on size (polymerase chain reaction [PCR]/polyacrylamide gel electrophoresis [PAGE]). We describe a simple approach to the detection of clonal TCR-gamma gene rearrangement using temporal temperature gradient gel electrophoresis (TTGE) that uses a gradual and uniform increase in the temperature of a constant denaturing gel to resolve different DNA molecules based on base pair composition. We tested 42 clinical specimens (30 blood specimens and 12 formalin-fixed paraffin-embedded tissues) for T-cell clonality by PCR/PAGE and PCR/TTGE. Concordant results were obtained in only 22 specimens (52%). Of the 20 discordant cases, 18 samples were positive by TTGE and negative by PAGE. For all of the discordant cases, the TTGE yielded results that correlated better with the clinical data than did the PAGE method. We conclude that PCR/TTGE is more accurate and easier to perform than current methods for detecting clonal populations of T cells. The T-cell receptor (TCR) gene rearrangement provides a convenient genetic marker for demonstrating clonality in T- cell malignant neoplasms. During normal T-cell development, each TCR gene (alpha, beta, gamma, and delta) can rearrange, leading to highly diverse TCR proteins. Among the 4 TCR gene types, the TCR-gamma genes are the first to rearrange. During the rearrangement of TCR-gamma, one of its different variable (V) regions combines with one of its different joining (J) regions. A few nucleotides (N region) are then inserted randomly into the VJ regions that are spliced together by terminal deoxynucleotidyl transferase, thereby rendering even higher sequence diversity in the TCR-gamma gene. It is believed that each mature T cell possesses an individual sequence of rearranged TCR-gamma gene. Therefore, analysis of TCR-gamma gene rearrangement is of practical value in determining the clonality of T-cell populations and for diagnosing T-cell malignant neoplasms. 1-5 To date, numerous techniques have been used to analyze TCR-gamma gene rearrangements. Southern blotting hybridization was the first widely used technique. 6 Because Southern blotting hybridization is time consuming and labor intensive and requires the use of large amounts of high-molecularweight DNA and radioactivity, polymerase chain reaction (PCR)-based techniques have become more popular in research and clinical laboratories. These techniques also permit the use of DNA extracted from formalin-fixed, paraffin-embedded specimens and are more rapid than hybridization approaches. 5,7-11 PCR amplification of the TCR-gamma gene from nonmalignant 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 American Society of Clinical Pathologists Am J Clin Pathol 2001;116:

2 Zhu et al / DETECTION OF CLONAL T-CELL RECEPTOR-GAMMA GENE REARRANGEMENT and junctional diversity in TCR-gamma gene rearrangement is limited compared with the diversity rendered by the randomly inserted N region, the amplified products may be of similar length. Thus, the limited diversity may result in false clonal bands when analyzed by standard gel electrophoresis methods that separate DNA molecules based solely on size. 5,10 This problem is known to occur when using either Southern blotting hybridization (which is based on agarose gel electrophoresis) or PCR followed by standard polyacrylamide gel electrophoresis (PAGE), which is currently used in most clinical laboratories. Furthermore, owing to the poor resolution of standard gel electrophoresis, DNA bands of low-frequency clones may be lost in the polyclonal smear background, leading to confusion in interpretation. To address these problems, electrophoresis techniques that resolve DNA molecules based on size and base pair composition have been explored. These techniques include single-strand conformation polymorphism (SSCP), 7 denaturing gradient gel electrophoresis (DGGE), 5,8,9 and temperature gradient gel electrophoresis (TGGE). 10 SSCP separates different DNA molecules based on their single-strand secondary structure conformations under certain electrophoretic conditions. Determined mainly by base composition, the conformation of DNA molecules can be changed as a result of base substitutions. DNA molecules with different conformations migrate at different rates on polyacrylamide gel and, therefore, can be separated from each other. To maximize the resolution of SSCP, more than 1 electrophoretic condition is usually needed. 12 DGGE resolves DNA molecules with differences of as little as a single base pair substitution. It is based on the melting properties of each DNA molecule, which are determined by its base-pair composition. Although DGGE has been gaining popularity to determine the clonality of T-cell populations in small or sparsely infiltrated specimens such as skin biopsy specimens, 5,8,9 the difficulty in preparing denaturing gradient polyacrylamide gel limits the routine use of this technique in clinical laboratories. TGGE is based on the same principle as DGGE but uses a temperature gradient along the length of a constant denaturing gel instead of a denaturant gradient. 10 We have studied an alternative approach to the detection of clonal TCR-gamma gene rearrangement using PCR followed by temporal temperature gradient gel electrophoresis (TTGE). 13,14 In this approach, DNA is separated by electrophoresis in a constant denaturing gel that is gradually and uniformly heated to achieve the effect of a denaturing gradient. A similar procedure was first described by Alkan and colleagues 15 but labeled as TGGE in that report. The gel for this procedure is much easier to prepare than true denaturing gradient gels in DGGE, and the TTGE system (~$4,000) is approximately 55% to 60% less expensive than a comparable TGGE system ($9,000-$10,000). In addition, it is much easier to calibrate a real temperature gradient in a TTGE system than a virtual temperature gradient in a TGGE system for the purpose of quality control in clinical operations. Herein we report the results of a comparative analysis of 42 clinical samples that were tested for clonal TCRgamma gene rearrangement by the PCR/PAGE and PCR/TTGE methods. We also describe some other properties of the assay such as its clinical accuracy, sensitivity, reproducibility, and stability. Materials and Methods Clinical Specimens The study set consisted of 42 consecutively submitted clinical specimens (30 peripheral blood specimens and 12 formalin-fixed, paraffin-embedded skin biopsies and lymph node tissues) that were referred to Quest Diagnostics, San Juan Capistrano, CA, for T-cell clonality testing. The clinical diagnosis for each sample was not available at the time of sample analysis. After the samples were tested by both methods, we contacted the referring physicians to obtain clinical correlation and follow-up data on their patients for up to 12 months following sample submission. DNA Extraction DNA from peripheral blood and formalin-fixed, paraffin-embedded tissues was purified using standard methods. In brief, for peripheral blood samples, a WBC pellet was prepared through Histopaque (Sigma Chemical, St Louis, MO) centrifugation and extracted with 1 ml of DNAzol (Molecular Research Center, Cincinnati, OH) at room temperature for 5 minutes. The supernatant was collected, mixed with 0.5 ml of ethanol, and incubated at room temperature for 5 minutes. After centrifugation, the DNA pellet was washed twice with 75% ethanol, air dried, and dissolved in 200 µl of double-distilled H 2 O. For formalin-fixed tissues, three to ten 10-µm-thick sections were prepared for each sample based on the sample size, digested with an appropriate amount of extraction buffer (50- mmol/l concentration of tris(hydroxymethyl)aminomethane (Tris); 2-mmol/L concentration of EDTA; 0.5% polysorbate 20; and 200 µl/ml of Proteinase K) at 56 C for 3 hours, and then incubated at 96 C for 10 minutes to inactivate the Proteinase. The supernatant was collected through centrifugation and extracted using an equal volume of phenol/chloroform. DNA was precipitated by mixing the aqueous phase with 1/20 volume of a 5-mol/L concentration of sodium chloride and 2.5 volume of ethanol and incubating at 20 C for 30 minutes. After centrifugation, the DNA pellet was 528 Am J Clin Pathol 2001;116: American Society of Clinical Pathologists

3 Hematopathology / ORIGINAL ARTICLE obtained, air dried, and dissolved in an appropriate amount of double-distilled H 2 O. The DNA samples extracted from blood were quantified using a spectrophotometer, and the concentrations were adjusted to approximately 200 ng/µl. Polymerase Chain Reaction Each DNA sample was amplified by PCR with HLA primers to serve as an internal control to verify successful DNA isolation and the presence of amplifiable DNA. PCR for TCR-gamma and HLA was performed in a 55-µL volume containing 5 µl of extracted DNA, 1 PCR Buffer II (Roche Molecular Systems, Branchburg, NJ), a 2-mmol/L concentration of magnesium chloride, 200-µmol/L concentrations of each deoxynucleoside triphosphate, 1.25 U of AmpliTaq DNA polymerase (Roche Molecular Systems), and a 227-nmol/L concentration of each of 2 primers. Rearranged TCR-gamma genes were amplified using primers Vgamma1-8 (5 -AGGGTTGTGTTGGAATCAGG- 3 ), Vgamma9 (5 -TAAATTCCAAATTCTTGGTTTA-3 ), Vgamma10 (5 -CTC AAC AAA ATC CGC AGC TCG ACG CAG CA-3 ), Vgamma11 (5 -CAA TCT CTG CTC AAG ATT GCT CAG GTG GG-3 ), or Vgamma12 (5 - ACT CTG CAG CCT CTT GGG CAC TGC TCT AAA-3 ) for the V region and Jgamma1/2 (5 -CGCCCGCCGCGCC- CCGCGCCCGTCCCGCCGCCCCCCTGTTC-CACTGC- CAAAGAGTTTCTT-3 ) for the J region. These primer sets were selected to amplify the most common VJgamma recombination types and to facilitate the method comparison between PCR/PAGE and PCR/TTGE. The underlined sequence is a GC-clamp region that was designed to introduce a high melting point domain at one end of the PCR amplicon to facilitate analysis by TTGE. The primers for the HLA gene (internal positive control) were GH26 (5 - GTGCTGCAGGTGTAAACTTGTACCAG-3 ) and GH27 (5 -CACGGATCCGGTAGCAGCGGTAGAGTTG-3 ). The amplification started with incubation at 95 C for 5 minutes followed by 10 cycles, each composed of 95 C for 1 minute, 60 C for 1 minute, and 72 C for 1 minute; then 25 cycles, each composed of 95 C for 20 seconds, 55 C for 30 seconds, and 72 C for 20 seconds; and finally elongation at 72 C for 10 minutes. Gel Electrophoreses The success of the PCR reaction was verified by running 10 µl of the TCR-gamma and HLA PCR products for each sample on a 2% agarose gel. Ten microliters of the amplified TCR-gamma gene product were mixed with an equal amount of loading buffer (0.05% bromophenol blue, 0.05% xylene cyanol, 70% glycerol) and analyzed by 8% Novex mini polyacrylamide gel (PAGE; Invitrogen, Carlsbad, CA) and by TTGE. For PAGE, the samples were run for 1 hour at 90 V. TTGE analysis used the DCode System (Bio-Rad Laboratories, Hercules, CA). For TTGE, the samples were electrophoresed for 6 hours at 90 V on an 8% polyacrylamide gel (37.5:1) containing a 1.75-mol/L concentration of urea and 10% formamide in 1 TAE buffer (40-mmol/L concentration of Tris base, 20-mmol/L concentration of glacial acetic acid, 1- mmol/l concentration of EDTA, ph 8.0). The optimum electrophoretic temperatures for the PCR products were determined using WinMelt DNA melting profile analysis software (Bio-Rad Laboratories). In the present study, the temperature was uniformly increased from 60 C to 66 C at a ramp rate of 1 C per hour. Gels were removed, stained with ethidium bromide (1 µg/ml) for 15 minutes, destained with water for 1 minute, and photographed under UV light using the ChemiImager 4400 Alpha Imager (Alpha Innotech, San Leandro, CA). PCR Amplicon Cloning The migration of a DNA molecule on TTGE depends on its thermal stability, which is determined mainly by its basepair composition instead of its length alone. Less stable molecules move more slowly. DNA molecules in conventional size markers may or may not contain high melting point temperature domains like the TCR-gamma gene PCR products in this study. Therefore, they may or may not be completely denatured during TTGE that is performed over a range of high temperatures. We observed that the bands of the 100-base-pair (bp) DNA size marker conventionally used for PAGE analysis did not migrate in a consistent manner under the temperatures used in TTGE (data not shown). Therefore, the conventional size markers could not be used as a migration reference to reflect the relative mobility of DNA molecules run on different gels at different times. To prepare a DNA migration marker as a reference to reflect the relative mobility of DNA molecules run on TTGE gels, the TCR-gamma gene PCR amplicons from a mixture of 10 samples positive for clonal T-cell rearrangement were ligated into pcr 2.1-TOPO cloning vector (Invitrogen). The recombinant vector DNA was then transformed into TOP10F (Invitrogen, Carlsbad, CA) Escherichia coli host bacteria. The transformed bacteria solution was spread on selective LB-agar plate (LB-Medium, B101, Vista, CA) with ampicillin and X-Gal (Promega, Madison, WI). After overnight incubation at 37 C, white colonies (with DNA inserts in the vector) were selected randomly and cultured in LB medium containing ampicillin for 6 hours. Plasmid DNA was isolated from each culture using a QIAprep Miniprep Kit (QIAGEN, Valencia, CA) and dissolved in doubledistilled H 2 O. TCR-gamma gene was amplified from each DNA clone and analyzed through agarose gel electrophoresis and then through TTGE to determine the nature of the insert. American Society of Clinical Pathologists Am J Clin Pathol 2001;116:

4 Zhu et al / DETECTION OF CLONAL T-CELL RECEPTOR-GAMMA GENE REARRANGEMENT A positive standard ladder for TTGE analysis was made by mixing DNA of 3 clones, amplicons of which showed apparently different migration rates on TTGE gel, and normal genomic DNA with proper proportions. The positive standard ladder DNA was used as a PCR amplification control, and its amplicon, a mixture of 3 DNA molecules with distinct thermal properties, was used as a migration marker for TTGE analysis. Assay Sensitivity, Specificity, Reproducibility, and Stability The lower limit of detection (sensitivity) of the PCR/TTGE assay for clonal TCR-gamma gene rearrangement was tested using cloned TCR-gamma gene standard DNA serially diluted 10-fold in human normal genomic DNA. The concentration of the positive standard DNA was measured by a spectrophotometer, and the DNA molecule copy number per microliter was calculated based on the theoretical molecular weight of the DNA clone (the cloning vector + the target insert 2,480,000). About 10 8, 10 7, 10 6, 10 5, 10 4, 10 3, 10 2, and 0 standard DNA molecules were spiked into 10 7 human normal genomic DNA (~1 µg) to form clonal TCR-gamma gene/normal genomic DNA (P/N) ratios of 10 1, 10 0, 10 1, 10 2, 10 3, 10 4, 10 5, and 0, respectively. The mixed DNA samples then were amplified and analyzed using the PCR/TTGE method. To compare the specificity of the PCR/PAGE with PCR/TTGE for detecting clonal TCR-gamma rearrangements, 1 positive sample DNA was serially diluted 2-fold with negative standard DNA. The diluted samples then were amplified by PCR and analyzed by both PAGE and TTGE. Intra-assay variability and interassay variability were evaluated by testing 10 clinical samples in triplicate and by testing the same 3 samples 3 times on different days. The sample stability study was carried out by testing blood samples stored at different temperature conditions (~4 C and room temperature) for different periods (1-7 days). A B M N P PSL N P Image 1 Detection of clonal T-cell receptor (TCR)-gamma gene rearrangement in clinical samples by polymerase chain reaction (PCR)/polyacrylamide gel electrophoresis (PAGE) (A) and PCR/temporal temperature gradient gel electrophoresis (TTGE) (B). PCR/PAGE displayed ambiguous results. Lanes 1, 3, 4, 5, and 7, samples showing clonal band patterns on TTGE; lanes 2, 6, 8, and 9, samples showing multiple faint bands on TTGE. The sample in lane 1 showed 4 clonal bands that may represent biallelic TCR-gamma gene rearrangement at the analyzed regions or multiclonal evolution or 1 strong rearrangement with 3 oligoclonal bands. M, 100-base-pair DNA size marker; N, negative standard DNA from a benign hyperplastic lymph node; P, positive standard DNA; PSL, positive standard ladder. Results Aliquots of PCR products first were analyzed on an agarose gel. PCR amplification for the HLA gene showed a single band of 242 bp for all 42 specimens (data not shown). Amplification with TCR-gamma gene specific primers showed a single band of approximately 200 bp for Vgamma1-8 + Jgamma1/2 amplicons, which then were analyzed using PAGE and TTGE Image 1. PAGE analysis of the PCR products showed that 10 of the 42 samples amplified with Vgamma1-8 + Jgamma1/2 primers yielded a single distinct band of approximately 200 bp superimposed on a background smear. These samples were presumed to contain monoclonal T cells. The other 32 samples yielded either a smear or a cluster of PCR products mixed with a background smear of a similar intensity. These samples were classified as containing polyclonal T cells. TTGE analysis of the PCR products resulted in 2 distinct patterns. The first pattern consisted of multiple faint bands, mostly at the top of the gel, with no predominant band evident. This pattern was characteristic of rearranged V-J fragments amplified from polyclonal T-cell populations without a predominant clone. Of the 42 samples tested, 16 showed such a pattern. The second pattern was 1 or more discrete bands that were much brighter than the multiple faint bands evident in the rest of the gel. These cases represented a clonally rearranged TCR-gamma gene, presumably in a background of polyclonal rearrangements. Of the 42 samples, 14 amplified with Vgamma1-8 + Jgamma1/2 primers showed clonal rearrangements of TCRgamma gene (Image 1). TTGE analysis also was performed on the PCR products of primer sets Vgamma9, 10, 11, Am J Clin Pathol 2001;116: American Society of Clinical Pathologists

5 Hematopathology / ORIGINAL ARTICLE PSL N Table 1 Comparison Between PCR/PAGE and PCR/TTGE * PCR/TTGE PCR/PAGE Positive Negative Total Positive Negative Total PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; TTGE, temporal temperature gradient gel electrophoresis. * Concordance, 52%; discrepancy, 48%. Image 2 Clonal T-cell receptor-gamma gene rearrangement between Vgamma11 and Jgamma1/2. Lanes 3, 6, 8, 10, and 12, samples showing clonal band patterns on temporal temperature gradient gel electrophoresis (TTGE); lanes 1, 2, 4, 5, 7, 9, and 11, samples showing absence of clonal T cells on TTGE. The sample in lane 12 showed 2 predominant bands. N, negative standard DNA; PSL, positive standard ladder. Jgamma1/2. Clonal bands were found for primer sets Vgamma11 + Jgamma1/2 in 22 of the 42 samples Image 2. A single distinct band was observed in 20 of 22 samples; 10 of these cases also yielded a single band with primer set Vgamma1-8 + Jgamma1/2. Two bands were observed in the remaining 2 samples with primer set Vgamma11 + Jgamma1/2 (lane 12 in Image 2). No clonal bands were observed in our study samples when using Vgamma9, Vgamma10, or Vgamma11 + Jgamma1/2. Thus, 26 of 42 samples were positive for clonal TCR-gamma gene rearrangement. Most samples with predominant clones displayed a single bright band for the Vgamma + Jgamma1/2 primer sets. Such a single band reflects monoallelic VJgamma rearrangement at the analyzed V and J locations. One sample amplified with Vgamma1-8 + Jgamma1/2 primers showed 2 discrete bands of rearranged TCR-gamma gene amplicon. These reflected biallelic VJgamma rearrangement with 2 homoduplexes. One sample showed 4 discrete bands that possibly represented another form of biallelic VJgamma rearrangement with 2 heteroduplexes at the upper position and 2 homoduplexes at the lower position. Alternatively, this case could represent multiclonal evolution or a single strong rearrangement with 3 oligoclonal bands. As shown in Table 1, comparison of the 2 methods showed that 8 samples positive by PCR/PAGE also were positive by PCR/TTGE and 14 samples negative by PCR/PAGE also were negative by PCR/TTGE (52% concordance). In addition, 2 samples positive by PCR/PAGE were negative by PCR/TTGE and 18 samples negative by PCR/PAGE were positive by PCR/TTGE (48% discordance). Of the 18 cases negative with PCR/PAGE and positive with PCR/TTGE, 17 were subsequently identified as having a T-cell neoplasm based on clinical and pathologic findings (peripheral T-cell lymphoma, 12 cases; cutaneous T-cell lymphoma, 5 cases; and prolymphocytic leukemia, 1 case). The other patient showed persistent skin rashes suggestive of a cutaneous T-cell lymphoma. The 2 patients whose samples were positive with PCR/PAGE and negative with PCR/TTGE did not show overt evidence of malignant neoplasm at 1 year after analysis. The sensitivity study showed that PCR/TTGE could detect clonal TCR-gamma gene at P/N ratios as low as 10 2, which is equivalent to 1 malignant T cell among 100 normal cells. This result was similar to that reported by Theodorou et al, 5 who tested DNA from the Jurkat T-cell line with a biallelic Vgamma1-8 + Jgamma1/2 and Vgamma11 + Jgamma1/2 rearrangements diluted in DNA from normal skin. Using the PCR/DGGE method, they reported a detectable signal at a 1% to 0.1% dilution range. 5 In the comparison of specificity between PCR/PAGE and PCR/TTGE Image 3, the negative standard showed no band on TTGE gel, while the serially diluted samples showed progressively decreased signals on TTGE gel with progressive dilutions. By contrast, on PAGE, the known negative standard showed a band of approximately 200 bp, which was difficult to distinguish from the bands in the positive samples, while the serially diluted samples did not show significant change in signal intensity. The results suggest that PCR/PAGE is difficult to interpret and more likely to yield inaccurate results. The results from the intra-assay and interassay variability studies did not show significant variability of the band pattern and signal intensity. The sample stability study showed that DNA extracted from samples stored at 4 C for up to 7 days and at room temperature for up to 4 days can be successfully used for PCR-TTGE without significant variability of the band pattern and signal intensity. American Society of Clinical Pathologists Am J Clin Pathol 2001;116:

6 Zhu et al / DETECTION OF CLONAL T-CELL RECEPTOR-GAMMA GENE REARRANGEMENT A M N N M N Image 3 Method comparison between polymerase chain reaction (PCR)/polyacrylamide gel electrophoresis (PAGE) and PCR/temporal temperature gradient gel electrophoresis (TTGE) using a sample positive for clonal T-cell receptorgamma gene rearrangement, serially diluted 2-fold with negative standard DNA. A, PCR/PAGE result. B, PCR/TTGE result. Lanes 1-6, a positive sample, diluted 2-, 4-, 8-, 16-, 32-, and 64-fold, respectively, with negative standard DNA. On the PAGE gel, the negative DNA PCR product showed an apparent clonal band, and the serially diluted samples did not show significant changes in signal intensity, suggesting that PCR/PAGE is likely to give inaccurate results. M, 100-basepair DNA size marker; N, known negative standard DNA; PSL, positive standard ladder. Discussion Several reports showed that clonal T-cell populations can be detected in certain benign conditions such as lymphomatoid papulosis and pityriasis lichenoides acuta 16 and in the blood of some elderly patients, 17 suggesting that T-cell clonality by itself may not be diagnostic of T-cell malignant neoplasms. However, since T-cell clonality is an essential feature of T-cell malignancy, assays for T-cell clonality are very helpful to distinguish lymphoma from reactive lymphoproliferation. These assays have been underused owing to difficulties in methods and interpretation of results. Size-based electrophoresis techniques give poor separation that fails to resolve TCR gene amplicon molecules with the same length but different base-pair compositions. Consequently, they sometimes give false-positive results. 5,8-10 Even samples known to be negative often may display a light band (Images 1 and 3) that may be a mixture of different TCR gene molecules with the same length or nonspecific PCR noise. For samples with relatively low proportions of monoclonal T cells, the determination of positivity depends on a comparison of band intensities between the sample and the negative control. Since the band intensity varies with several factors, including sample load variations, a subjective determination based on band intensity is likely to give inaccurate results. B Composition-based DNA electrophoresis techniques, which originally were used for gene mutation detection and screening, have been applied to detection of clonal TCRgamma gene rearrangements. These are thought to be superior to size-based methods with regard to sensitivity and specificity. 5,8-10 The complexity of these techniques, however, has hindered their use for routine testing in clinical laboratories. TTGE has been introduced to detect DNA mutations because it can resolve DNA molecules differing in as little as a single base pair substitution. 13,14 Its simplicity in gel preparation makes it a good candidate for application in clinical laboratory tests. The present study demonstrates that the PCR/TTGE approach for detection of clonal TCRgamma gene rearrangement is more accurate and easier to interpret than the PCR/PAGE method, which is the most widely used method in clinical laboratories. For TTGE, the critical parameters include concentrations of denaturants (urea and formamide), starting and ending temperatures, temperature ramp rate, and running time. All of these needed to be optimized first by computer modeling and then confirmed empirically to achieve maximal separation based on the thermal properties of the DNA molecule (refer to the Materials and Methods section). In this report, we describe the conditions that we believe yield optimal results for the primer sets we used. TCR-gamma gene rearrangement involves recombination between one of the V regions (Vgamma1-8, Vgamma9, Vgamma10, and Vgamma11) and one of the J regions (Jgamma1/2 and JgammaP1/2). In this study, we used primers Vgamma1-8, 9, 10, 11, and 12 for the V region and Jgamma1/2 for the J region to analyze the most common VJgamma recombinations. Our results clearly indicate that optimum detection of T-cell neoplasms requires the use of multiple primer sets for the Vgamma region. It is also likely that using additional J region primers would have increased the yield of positive results still further, although this study was not specifically intended to investigate this possibility or to determine the best combination of primers for this assay. Among the 14 samples that were positive by PCR/TTGE using the Vgamma1-8 + Jgamma1/2 primers, 2 samples showed multiple bands (1 with 2 and 1 with 4 bands), and 12 showed a single predominant TCR-gamma gene. Ten of the cases with a single band subsequently were found to have another type of VJgamma rearrangement when analyzed using the Vgamma11 + Jgamma1/2 primer set, which detects rearrangements between Vgamma11 and Jgamma1/2 regions (data not shown). This finding suggested that at least 86% (12/14) of the clonal VJgamma rearrangements in our study were biallelic heterozygosity, ie, the predominant clonal T cells carried 2 different TCR-gamma gene alleles with different VJgamma rearrangements. Similar results have been described by others. 5,10 It is unclear 532 Am J Clin Pathol 2001;116: American Society of Clinical Pathologists

7 Hematopathology / ORIGINAL ARTICLE whether the heterozygosity at TCR-gamma gene locus is a common phenomenon among normal T-cell populations, which further diversifies TCR-gamma structure for each T- cell subpopulation. Multiple bright bands found on a TTGE gel should be interpreted with caution since they may be due to a number of causes. One possibility is that the bands are due to biallelic VJgamma rearrangement between Vgamma and Jgamma regions, resulting in 2 heteroduplexes and 2 homoduplexes or 2 homoduplexes only. The former is a typical pattern for 2 different DNA molecules with minor base pair substitution(s), while the latter may reflect 2 markedly different types of DNA molecules that are too dissimilar to form heteroduplex structures. In that case, 2 bands should display similar signal intensity. Alkan and colleagues 15 showed that the MOLT-16 cell line shows a biallelic rearrangement pattern when analyzed by TTGE. Similar findings have been reported by others using the DGGE method. 5,8 Another possibility is that the specimen contains a multiclonal T-cell evolution or a single strong rearrangement with multiple oligoclonal bands. Finally, it is possible that nonspecific PCR amplification under certain circumstances could result in artificial DNA bands. Owing to the high sensitivity and resolution of TTGE, multiple faint bands are often visible, even in tissue samples such as a negative control without a predominant T-cell clone. These bands, however, are very faint in comparison with the strong TCR-gamma gene clonal bands, which migrate at different positions. Such artificial bands should be disregarded to avoid false-positive results. Like other DNA composition-based electrophoretic separation methods, 5,7,10 TTGE gives reproducible band migration patterns for the same DNA molecules, as shown in our interassay and intra-assay variability studies. Therefore, this technique potentially can be used to compare multifocal lesions from a single patient or to compare a recurrent lesion with a primary one in a patient previously diagnosed with a T-cell malignant neoplasm. Theodorou and colleagues 5 reported that TCR-gamma rearrangement remained unchanged in different specimens simultaneously or subsequently collected from the same patients using PCR/DGGE. Murphy and colleagues 7 also detected identical gel banding patterns by PCR/SSCP in serial skin biopsies from the same patients, indicating that the same neoplastic clone was present during the course of the disease. Our findings confirm and significantly extend the observations previously reported by Alkan and colleagues, 15 who compared a TTGE method (labeled as TGGE in their report) with Southern blotting for detection of TCR gene rearrangements in clinical specimens and in a MOLT-16 cell line. In our study, we compared the TTGE method with the PAGE method and with the clinical pathologic diagnosis for each patient. Although there are some technical differences between the study of Alkan et al 15 and our study (for example, different primer sets, different temperature gradients, multiplex vs regular PCR), we also conclude that TTGE is a superior method for detecting clonal rearrangements of T cells. In addition, we showed that the TTGE assay is highly reproducible, especially when interpreted in conjunction with the positive standard ladder that we describe in this report. PCR/TTGE analysis of the rearranged TCR-gamma gene is a simple, accurate, and sensitive technique to diagnose and monitor patients with T-cell malignant neoplasms. The sequence-specific imprint of TCR-gamma gene PCR products by TTGE also provides a potentially useful tool for evaluation of multifocal or recurrent T-cell lesions in individual patients. From the 1 Nichols Institute, Quest Diagnostics, San Juan Capistrano, CA; and 2 Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. Presented in part at the American Society of Hematology Annual Meeting, San Francisco, CA, December Address reprint requests to Dr Samoszuk: Dept of Hematology/Oncology, Nichols Institute, Quest Diagnostics, Ortega Highway, San Juan Capistrano, CA. References 1. Flug F, Pelicci PG, Bonett F, et al. T-cell receptor gene rearrangements as markers of lineage and clonality in T-cell neoplasms. Proc Natl Acad Sci U S A. 1985;82: Yanagi Y, Yoshikai Y, Leggett K, et al. A human T cell specific cdna clone encodes a protein having extensive homology to immunoglobulin chains. Nature. 1984;308: Waldmann TA, Davis MM, Bongiovanni K, et al. Rearrangements of genes for the antigen receptor on T cells as markers of lineage and clonality in human lymphoid neoplasms. N Engl J Med. 1985;313: Raulet DH. The structure, function, and molecular genetics of the gamma/delta T cell receptor. Annu Rev Immunol. 1989;7: Theodorou I, Delfau MH, Bigorgne C, et al. Cutaneous T-cell infiltrates: analysis of T-cell receptor gamma gene rearrangement by polymerase chain reaction and denaturing gradient gel electrophoresis. Blood. 1995;86: Spagnolo DV, Taylor J, Carrello S, et al. Southern blot analysis of lymphoproliferative disorders: use and limitations in routine surgical pathology. Pathology. 1994;26: Murphy M, Signoretti S, Kadin ME, et al. Detection of TCRgamma gene rearrangements in early mycosis fungoides by non-radioactive PCR-SSCP. J Cutan Pathol. 2000;27: Anderson WK, Li N, Bhawan J. Polymerase chain reaction denaturing gradient gel electrophoresis (PCR/DGGE)-based detection of clonal T-cell receptor gamma gene rearrangements in paraffin-embedded cutaneous biopsies in cutaneous T-cell lymphoproliferative diseases. 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