A Novel Method for Interpretation of T-Cell Receptor γ Gene Rearrangement Assay by Capillary Gel Electrophoresis Based on Normal Distribution
2007; Elsevier BV; Volume: 9; Issue: 1 Linguagem: Inglês
10.2353/jmoldx.2007.060032
ISSN1943-7811
AutoresFrank C. Kuo, Dimity Hall, Janina A. Longtine,
Tópico(s)Viral-associated cancers and disorders
ResumoT-cell receptor gamma (TRG) gene rearrangement status is useful for the differential diagnosis of T-cell lesions. The BIOMED-2 protocol that uses two sets of Jγ and four sets of Vγ primers in a multiplex, two-tube reaction followed by capillary gel electrophoresis is emerging as a standard assay for this application. Here, we report a computer-aided method to evaluate the significance of a peak in this TRG clonality assay. A best-fit normal distribution (ND) curve and the χ2 error for each peak are used to determine whether a peak is significantly taller than the background (cutoff for Vγ1–8 is 1). Eighty clinical samples that have been previously analyzed by a GC-clamped primer polymerase chain reaction/denaturing gradient gel electrophoresis assay were reanalyzed with the BIOMED-2 assay and scored by the ND method and four previously published methods: relative peak height (RPH), relative peak ratio (RPR), height ratio (HR), and peak height ratio (Rn). A greater than 90% concordance rate was observed between RPH and ND analysis, whereas RPR, Rn, and HR had a lower threshold to call a peak positive. The advantage of the ND method is that it is more objective, reproducible, and can be automated. T-cell receptor gamma (TRG) gene rearrangement status is useful for the differential diagnosis of T-cell lesions. The BIOMED-2 protocol that uses two sets of Jγ and four sets of Vγ primers in a multiplex, two-tube reaction followed by capillary gel electrophoresis is emerging as a standard assay for this application. Here, we report a computer-aided method to evaluate the significance of a peak in this TRG clonality assay. A best-fit normal distribution (ND) curve and the χ2 error for each peak are used to determine whether a peak is significantly taller than the background (cutoff for Vγ1–8 is 1). Eighty clinical samples that have been previously analyzed by a GC-clamped primer polymerase chain reaction/denaturing gradient gel electrophoresis assay were reanalyzed with the BIOMED-2 assay and scored by the ND method and four previously published methods: relative peak height (RPH), relative peak ratio (RPR), height ratio (HR), and peak height ratio (Rn). A greater than 90% concordance rate was observed between RPH and ND analysis, whereas RPR, Rn, and HR had a lower threshold to call a peak positive. The advantage of the ND method is that it is more objective, reproducible, and can be automated. Polymerase chain reaction (PCR) amplification of gene segments for the rearranged antigen receptors [T-cell receptor (TCR) β (TRB)] and γ (TRG) chains and immunoglobulin (Ig) heavy (IGH) and κ light chains (IGK) by fluorescently labeled primers, followed by capillary gel electrophoresis (CE)1Miller JE Wilson SS Jaye DL Kronenberg M An automated semiquantitative B and T cell clonality assay.Mol Diagn. 1999; 4: 101-117Crossref PubMed Scopus (30) Google Scholar,2Simon M Kind P Kaudewitz P Krokowski M Graf A Prinz J Puchta U Medeiros LJ Sander CA Automated high-resolution polymerase chain reaction fragment analysis: a method for detecting T-cell receptor gamma-chain gene rearrangements in lymphoproliferative diseases.Am J Pathol. 1998; 152: 29-33PubMed Google Scholar has increasingly been used as an ancillary diagnostic test to assess T- and B-cell clonality.3Greiner TC Raffeld M Lutz C Dick F Jaffe E Analysis of T cell receptor γ gene rearrangements by denaturing gradient gel electrophoresis of GC-clamped polymerase chain reaction products.Am J Pathol. 1995; 146: 46-55PubMed Google Scholar,4Theodorou I Bigorgne C Delfau MH Lahet C Cochet G Vidaud M Raphael M Gaulard P Farcet JP VJ rearrangements of the TCR gamma locus in peripheral T-cell lymphomas: analysis by polymerase chain reaction and denaturing gradient gel electrophoresis.J Pathol. 1996; 178: 303-310Crossref PubMed Google Scholar The test is based on the realization that during the assembly of the TCR and Ig genes, the length and the sequence content of the rearranged genes are highly polymorphic such that each B cell and T cell has its unique set of antigen receptor genes. If a reactive tissue is sampled, the distribution of the sizes of the investigated segment is expected to follow a normal (Gaussian) distribution (Figure 1A). If a clonal population is present, a fragment of a distinct size can be expected to predominate in the product mixture (Figure 1B). There is a plethora of published reports with different primer designs and experimental protocols exploiting this principle of clonality analysis.5van Dongen JJ Langerak AW Bruggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E Garcia-Sanz R van Krieken JH Droese J Gonzalez D Bastard C White HE Spaargaren M Gonzalez M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98–3936.Leukemia. 2003; 17: 2231-2257Google Scholar6Hodges E Williams AP Harris S Smith JL T-cell receptor molecular diagnosis of T-cell lymphoma.Methods Mol Med. 2005; 15: 197-215Google Scholar7Beaubier NT Hart AP Bartolo C Willman CL Viswanatha DS Comparison of capillary electrophoresis and polyacrylamide gel electrophoresis for the evaluation of T and B cell clonality by polymerase chain reaction.Diagn Mol Pathol. 2000; 9: 121-131Crossref PubMed Scopus (43) Google Scholar8Sprouse JT Werling R Hanke D Lakey C McDonnel L Wood BL Sabath DE T-cell clonality determination using polymerase chain reaction (PCR) amplification of the T-cell receptor gamma-chain gene and capillary electrophoresis of fluorescently labeled PCR products.Am J Clin Pathol. 2000; 113: 838-850Crossref PubMed Scopus (42) Google Scholar9Krafft AE Taubenberger JK Sheng ZM Bijwaard KE Abbondanzo SL Aguilera NS Lichy JH Enhanced sensitivity with a novel TCRgamma PCR assay for clonality studies in 569 formalin-fixed, paraffin-embedded (FFPE) cases.Mol Diagn. 1999; 4: 119-133Crossref PubMed Scopus (33) Google Scholar Currently, two commercially available kits are most widely used by diagnostic laboratories in commercial facilities and academic medical centers. One5van Dongen JJ Langerak AW Bruggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E Garcia-Sanz R van Krieken JH Droese J Gonzalez D Bastard C White HE Spaargaren M Gonzalez M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98–3936.Leukemia. 2003; 17: 2231-2257Google Scholar,10Sandberg Y Heule F Lam K Lugtenburg PJ Wolvers-Tettero IL van Dongen JJ Langerak AW Molecular immunoglobulin/T-cell receptor clonality analysis in cutaneous lymphoproliferations. Experience with the BIOMED-2 standardized polymerase chain reaction protocol.Haematologica. 2003; 88: 659-670PubMed Google Scholar was developed by BIOMED-2, a European consortium, and the second was developed and marketed by InVivoScribe Technologies LLC (San Diego, CA) using proprietary primer sequences.11Oda RP Wick MJ Rueckert LM Lust JA Landers JP Evaluation of capillary electrophoresis in polymer solutions with laser-induced fluorescence detection for the automated detection of T-cell gene rearrangements in lymphoproliferative disorders.Electrophoresis. 1996; 17: 1491-1498Crossref PubMed Scopus (15) Google Scholar For B cells, both kits use three separate PCR reactions with consensus primers to the three framework regions of the IGH gene (FRI, FRII, and FRIII). For T cells, both have four distinct primers for the V segments (Vγ1–8, Vγ9, Vγ10, and Vγ11) and two primers for the J segments (Jγl.l, Jγ1.3, and Jγ2.1; and Jγ2.3) of the TRG gene that are fluorescently labeled with different fluorophores. For the IGH test, the interpretation of the CE result is usually more straightforward. The reactive samples have a range of product peaks at three-base intervals showing a normal distribution. When a clonal population is present, one or two distinct peaks are generally observed in addition to the reactive background. Because the consensus primers used for the three framework regions cover a large repertoire of the VDJ segments, the background amplification is more robust, and the same rearrangements can often be amplified in more than one reaction, providing easily obtainable interobserver concordance (data not shown). TRG reactions, on the other hand, frequently present interpretative challenges in clinical specimens because more than one prominent peak of varying heights is common in TRG amplifications, either with or without an accompanying recognizable polyclonal background (Figure 1C). In those specimens, it is difficult to reach interobserver consensus without objective criteria for interpretation. Four methods have been previously proposed to aid in the determination of the significance of a peak by calculating the ratio of the height of the peak in question to that of the polyclonal background.8Sprouse JT Werling R Hanke D Lakey C McDonnel L Wood BL Sabath DE T-cell clonality determination using polymerase chain reaction (PCR) amplification of the T-cell receptor gamma-chain gene and capillary electrophoresis of fluorescently labeled PCR products.Am J Clin Pathol. 2000; 113: 838-850Crossref PubMed Scopus (42) Google Scholar,12Lee SC Berg KD Racke FK Griffin CA Eshleman JR Pseudo-spikes are common in histologically benign lymphoid tissues.J Mol Diagn. 2000; 2: 145-152Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar13Greiner TC Rubocki RJ Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements.J Mol Diagn. 2002; 4: 137-143Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar14Luo V Lessin SR Wilson RB Rennert H Tozer C Benoit B Leonard DG Detection of clonal T-cell receptor gamma gene rearrangements using fluorescent-based PCR and automated high-resolution capillary electrophoresis.Mol Diagn. 2001; 6: 169-179PubMed Google Scholar These methods work well for experienced observers when a background amplification is clearly present but are less useful in cases where background amplifications are not robust. Three of these methods rely on determination of the background height, which can only be deciphered subjectively by the observer; thus reproducible measurements are more difficult to perform in practice. This is especially true in the subsets of reactions where the V segment usage is underrepresented, producing a small background. Here, we report a new method to evaluate the significance of a peak in a TRG clonality assay. The data are exported from the capillary gel instrument software (Applied Biosystems 3100xl GeneMapper v3.7; Foster City, CA) and fitted to a normal distribution curve by computer algorithms. Individual peaks are evaluated for their closeness to the fitted curve. Specimens where all data points can be successfully fitted to a normal distribution curve are considered to be reactive or nonclonal, and specimens with peaks having χ2 errors significantly deviated from the curve are interpreted as clonal. This method is objective as the curve fitting and calculation steps are fully computerized. It also takes into consideration the size of the individual peaks and theoretically can improve the sensitivity of tests when the clonal peak is at the edge of the expected size range for a given primer set compared with relative peak height (RPH). We performed titration experiments and used a training set of cases to select a cutoff value to separate clonal and reactive specimens. Using the cutoff value, we evaluated and compared the TRG gene rearrangement results of 80 cases of T-cell lesions analyzed by GC-clamped primers and denaturing gradient gel electrophoresis (DGGE)3Greiner TC Raffeld M Lutz C Dick F Jaffe E Analysis of T cell receptor γ gene rearrangements by denaturing gradient gel electrophoresis of GC-clamped polymerase chain reaction products.Am J Pathol. 1995; 146: 46-55PubMed Google Scholar with those by fluorescently labeled BIOMED-2 primers10Sandberg Y Heule F Lam K Lugtenburg PJ Wolvers-Tettero IL van Dongen JJ Langerak AW Molecular immunoglobulin/T-cell receptor clonality analysis in cutaneous lymphoproliferations. Experience with the BIOMED-2 standardized polymerase chain reaction protocol.Haematologica. 2003; 88: 659-670PubMed Google Scholar (InVivoScribe Technologies, LLC) and CE. The latter results were scored by five methods: RPH,12Lee SC Berg KD Racke FK Griffin CA Eshleman JR Pseudo-spikes are common in histologically benign lymphoid tissues.J Mol Diagn. 2000; 2: 145-152Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar relative peak ratio (RPR),13Greiner TC Rubocki RJ Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements.J Mol Diagn. 2002; 4: 137-143Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar height ratio (HR),8Sprouse JT Werling R Hanke D Lakey C McDonnel L Wood BL Sabath DE T-cell clonality determination using polymerase chain reaction (PCR) amplification of the T-cell receptor gamma-chain gene and capillary electrophoresis of fluorescently labeled PCR products.Am J Clin Pathol. 2000; 113: 838-850Crossref PubMed Scopus (42) Google Scholar peak height ratio (Rn),5van Dongen JJ Langerak AW Bruggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E Garcia-Sanz R van Krieken JH Droese J Gonzalez D Bastard C White HE Spaargaren M Gonzalez M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98–3936.Leukemia. 2003; 17: 2231-2257Google Scholar and our new method, normal distribution (ND). A 90% concordance rate is observed between RPH and ND analysis. The RPR, Rn, and HR methods have a lower threshold for peak calling and tend to overcall when compared with results obtained by DGGE and the RPH or ND methods. The advantage of the new method is that it is automated, more objective, and reproducible. Twenty-nine polyclonal samples (22 reactive tissue samples and seven specimens of peripheral blood lymphocytes) and 51 samples with clonal TRG gene rearrangements identified by DGGE and/or Southern blots were used to validate the new method (see also Table 1). The histological diagnosis of these 51 samples include 11 cases of peripheral T-cell lymphoma, 21 cases of mycosis fungoides, 14 cases of atypical lymphoid infiltrate, one case of anaplastic large cell lymphoma, one case of T-cell acute lymphoblastic leukemia, and three cases of T-cell large granular lymphocytic leukemia. Twenty-five cases were amplified from frozen tissue, nine cases from blood/bone marrow, and 56 cases from paraffin-embedded tissue. These clinical samples, which were sequentially accessioned in our lab over a 6-month period in 2004 (July to December), were previously tested by either Southern blot and/or DGGE. PEER and Jurkat T cell line DNA and human polyclonal control DNA are supplied with the TCRγ Gene Clonality Assay Kit (InVivoScribe Technologies, LLC).Table 1Features of Cases Used in the Evaluation of the New MethodHistologic diagnosis Peripheral T-cell lymphoma, NOS11 Mycosis fungoides21 Atypical lymphoid infiltrate14 Anaplastic large cell lymphoma1 Acute T-cell lymphoblastic lymphoma1 Large granular lymphocyte leukemia, T cell phenotype3 Reactive, nonneoplastic29Tissue type (excluding blood/marrow) Frozen25 Paraffin-embedded56Tissue source Blood/bone marrow9 Skin49 Lymph node18 Others14Vγ usages (by DGGE)*One case can have more than one rearranged allele detected. Vγ1–844 Vγ97 Vγ1012NOS, nitric-oxide synthase.* One case can have more than one rearranged allele detected. Open table in a new tab NOS, nitric-oxide synthase. DNA was extracted from fresh/frozen tissue with PureGene DNA Isolation kits (Gentra Systems, Minneapolis, MN) according to the manufacturer's instruction. Paraffin-embedded tissue was cleared of paraffin by xylene, followed by 100% ethanol rinse. The deparaffinized tissue was then processed with QIAGEN QIAamp DNA micro kits (QIAGEN 56304; QIAGEN, Valencia, CA). Serial twofold dilutions of tumor DNA into polyclonal human DNA were prepared with PEER and Jurkat DNA. Amplification of TRG gene by GC clamped primers followed by DGGE was performed according to methods described by Greiner et al.3Greiner TC Raffeld M Lutz C Dick F Jaffe E Analysis of T cell receptor γ gene rearrangements by denaturing gradient gel electrophoresis of GC-clamped polymerase chain reaction products.Am J Pathol. 1995; 146: 46-55PubMed Google Scholar When high-molecular weight DNA was available, the DNA was also digested with two or three restriction enzyme combinations and separated on 0.8% agarose gel and transferred to a nylon membrane. Jγ probes for TRG were radiolabeled with [32P]dCTP, hybridized to the membrane overnight, washed, and exposed to X-ray film according to standard Southern blot procedures. The T-cell clonality assay was performed according to manufacturer's instruction with reagents purchased from InVivoScribe Technologies LLC (TCRG Gene Clonality Assay for ABI Fluorescence Detection, InVivoScribe no. l-207-0021). The kit uses the primer sequences published by the BIOMED-25van Dongen JJ Langerak AW Bruggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E Garcia-Sanz R van Krieken JH Droese J Gonzalez D Bastard C White HE Spaargaren M Gonzalez M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98–3936.Leukemia. 2003; 17: 2231-2257Google Scholar and had all reagents needed except for sample DNA and Taq polymerase (AmpliTAQ Gold; Applied Biosystems). Two multiplex PCR reactions were performed for each specimen. Tube A contained primers that target the Vγ1–8 + Vγ10 sequences and Jγ1.1/2.1 and Jγ1.3/2.3. Tube B contained primers that target the Vγ9 + Vγ11 sequences and all Jγ1.1/2.1 and Jγ1.3/2.3. JγP primer was not used in this kit. A Control Size Ladder master mix was used in a third reaction. The PCR reactions were conducted in either a PE 9600 thermocycler (Perkin-Elmer, Foster City, CA) or an ABI 2700 thermocycler (Applied Biosystems) with 0.5 μg of DNA used in each reaction. The PCR program was composed of heating initially to 95°C for 7 minutes, followed by 35 cycles of 95°C for 45 seconds, 60°C for 45 seconds, 72°C for 90 seconds, plus a final extension of 72°C for 10 minutes. After PCR, 10 μl of HiDi formamide/Genescan 400HD ROX size standard mixture was mixed with 1 μl of PCR product, transferred to a 96-well plate, heated to 95°C for 3 minutes, snap-chilled for 5 minutes, and loaded onto a 3130xl Genetic Analyzer (ABI, Foster City, CA) for capillary gel electrophoresis. The raw data for each sample run were loaded into ABI GeneMapper v3.7 software for peak calling and measurement of peak size and area under the curve. All of the data on peak size, height, and area were exported as a comma-delimited file and imported into Microsoft Excel (Redmond, WA) for analysis. First, the expected size range data provided by BIOMED-2 were used to generate a population of idealized ND of peaks for each case. The height of each peak was then calculated for every given product size (expected, E). The observed (O) peak height at each size was then fitted into this idealized distribution by repeated iterations until the sum of χ2 error [Σ(O − E)2Simon M Kind P Kaudewitz P Krokowski M Graf A Prinz J Puchta U Medeiros LJ Sander CA Automated high-resolution polymerase chain reaction fragment analysis: a method for detecting T-cell receptor gamma-chain gene rearrangements in lymphoproliferative diseases.Am J Pathol. 1998; 152: 29-33PubMed Google Scholar/E] for all of the peaks reached a minimum (χ2 goodness-of-fit test).15Snedecor GW Cochran WG Statistical Methods. 8th ed. Iowa State University Press, Iowa City1989Google Scholar If no χ2 error was greater than 1, then the null hypothesis of a normal distribution for the dataset was accepted, and the specimen was declared polyclonal. If χ2 error of any of the peaks was greater than 1, then that data point was temporarily removed, and the process of curve fitting was repeated. This step mimics the situation where a clonal population is present in a mixture of reactive T cells, and if the clonal T cells are removed from the specimen, the remaining T cells can be expected to give rise to a normal distribution. This process may be repeated once more because neoplastic T cells may have two rearranged alleles. At the end of the process, the result of the analysis was shown in a maximum of eight separate charts, one for each V and J primer combination. A summary was also provided to highlight peaks with large χ2 errors. A computer program in Microsoft Visual Basic for Application (VBA) has been written and implemented as a macro that can be invoked by clicking an icon on the standard toolbar. This program is available on request. To compare the existing methods, the data were also examined to produce a value based on the RPH,12Lee SC Berg KD Racke FK Griffin CA Eshleman JR Pseudo-spikes are common in histologically benign lymphoid tissues.J Mol Diagn. 2000; 2: 145-152Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar RPR,13Greiner TC Rubocki RJ Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements.J Mol Diagn. 2002; 4: 137-143Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar HR,8Sprouse JT Werling R Hanke D Lakey C McDonnel L Wood BL Sabath DE T-cell clonality determination using polymerase chain reaction (PCR) amplification of the T-cell receptor gamma-chain gene and capillary electrophoresis of fluorescently labeled PCR products.Am J Clin Pathol. 2000; 113: 838-850Crossref PubMed Scopus (42) Google Scholar and Rn14Luo V Lessin SR Wilson RB Rennert H Tozer C Benoit B Leonard DG Detection of clonal T-cell receptor gamma gene rearrangements using fluorescent-based PCR and automated high-resolution capillary electrophoresis.Mol Diagn. 2001; 6: 169-179PubMed Google Scholar methods. In brief, the data were inspected by one of us (D.H.) to determine the maximal height of the polyclonal background (h0). The height of the peak of interest (h1) was read from the GeneMapper output. In cases where a reliable h0 could not be determined (discernable background distribution is not always present), we excluded such cases from this study. In the BIOMED-2 assay, the reactions were separate, and the product lengths were different. In the original publications, these methods were designed to analyze pooled PCR products from several individual V-J amplifications. We adapted these methods to each individual BIOMED-2 reaction. RPH was calculated as (h1 − h0)/h0, and RPR was calculated as h1/h0. HR is the same as RPR. Rn is the ratio between h1 and the average of the two immediate flanking peaks. Our initial aim was to try to fit the CE data into a computer-generated, idealized ND curve. This alone worked fairly well for cases that clearly fell into a normal distribution by visual inspection (cases with a pattern as in Figure 1A) but did not work well for cases that were clonal (Figure 1B) or were without a discernable polyclonal background (Figure 1C). In those situations, attempts to fit all of the data into a curve appeared to skew the best-fit curve toward the higher peaks. This is because most curve-fitting algorithms tend to be more weighted toward the higher peaks; as a result, the taller peaks are fitted onto a curve while the smaller peaks are ignored by the algorithm. Recognizing this fact, we next tried to remove the tallest outlier from the dataset before curve fitting and to see whether that would yield a more realistic normal distribution representing the "reactive" portion of the data. Because both rearranged alleles in a clonal T cell may be amplifiable by the same Vγ-Jγ primer set, we also factored in the situations where two outliers may need to be removed to identify the best-fit curve. After a best-fit curve was obtained with this modified dataset, all of the peaks in the original dataset could then be evaluated to determine whether each peak belongs on the curve by χ2 analysis. One or two peaks too tall to fit into the curve were considered as evidence of the presence of a clonal population. We tested this algorithm first by using DNA isolated from a reactive sample (human tonsil) in a standard BIOMED-2 TRG assay. As shown in Figure 2A, the peaks from polyclonal samples were fitted into an idealized ND curve created by the mean and SD of the known size range of each of the TRG primer combinations. This was true with all eight possible primer combinations in the BIOMED-2 test (only Vγ10 reactions are shown as examples). Next, we tested two clonal T-cell samples, one with a known Vγ1–8 and one with a known Vγ9 rearrangement. As expected, when all of the data points from the PCR amplifications were used, the curve that minimized the χ2 error had a total error too big to be accepted as a fit. When we removed the outlier peak and repeated the curve fitting, a best-fit curve that included most of the reactive peaks was successfully found for both cases (Figure 2B). Using the best-fit curve, we evaluated all of the peaks, and the clonal peaks in both cases gave χ2 errors more than 100-fold greater than the rest of the peaks. These initial data supported our notion that an objective method that quantitatively measures the extent of deviation of a peak from a normal distribution curve can be adapted to analyze the results from a TRG assay. Having refined our algorithm, we next determined whether cutoff values for χ2 error can be selected with acceptable sensitivity and specificity for evaluation of clinical samples. We first selected 10 clonal and 10 polyclonal clinical samples and used the data as a training set to identify a reasonable cutoff value. To facilitate this, we developed a computer program that automates the process of curve fitting and identification of the peaks with large χ2 errors. The program is written with VBA and runs on Microsoft Excel 2000 and above. It takes exported delimited files from GeneMapper 3.7 and outputs the result as individual charts for each TRG primer reaction and a summary table. The input files can be processed individually or as a batch. A flow diagram of this program is illustrated in Figure 3. Using this program, we analyzed the dataset of 20 samples and examined the χ2 errors of all of the peaks. Without exceptions, the 10 polyclonal samples gave no peak with a χ2 error greater than 0.08. The large peaks of the 10 clonal samples, on the other hand, had χ2 error values ranging from 1.05 to over 100. Based on this data, we tentatively set the cutoff for χ2 error at 1. Errors less than 0.1 are considered to be insignificant and those between 1 and 0.1 to be equivocal; although in this training set, we did not see any peaks with errors that fell within this range. Next, we PCR-amplified and analyzed T cell line (PEER) DNA serially diluted into tonsil DNA from a ratio of 1:1 down to 1:255 (50% down to 0.5% tumor) to refine the cutoff value of our method. For Vγ 1–8 reactions (Figure 4A), the χ2 error ranged from a high of 12.23 at 50% tumor to a low of 0.022 at 3% tumor. Surveying the χ2 errors of other nonclonal peaks in the reaction showed that more than 95% have a χ2 error of less than 0.05. At 1:7 dilution, which is equivalent to about 15% tumor cells, the χ2 error was 1.38. Based on this data, we selected a χ2 error of 1 as a cutoff to give us a specificity of 100% in our training set and a sensitivity of about 10% tumor cells, which is roughly twice as many cells as identified by the lower limit of detection of Southern blot assays. The results for Vγ9 and Vγ10 primer sets were distinctly different from those of the Vγ1–8. With the Vγ9 primer set, a 1:255 dilution yielded a χ2 error of 1, ie, as little as 0.5% clonal cells (Figure 4B). This is probably due to the underrepresentation of T cells in a mixed cell population that use Vγ9 in TRG rearrangement compared with Vγ1–8. We found similar results for Vγ10 reactions (data not shown). Thus, a uniform χ2 error cutoff for all V regions will not identify similar percentages of clonal cells. To further validate our algorithm and computer program, we took sequentially accessioned clinical samples over a 6-month period in 2004 (July to December) that were previously tested by the Southern blot and/or DGGE (see Materials and Methods) technique and amplified them by the BIOMED-2 kit. Of these, 80 samples were successfully amplified (Table 1) and were analyzed by four previously published methods, RPH,12Lee SC Berg KD Racke FK Griffin CA Eshleman JR Pseudo-spikes are common in histologically benign lymphoid tissues.J Mol Diagn. 2000; 2: 145-152Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar RPR,13Greiner TC Rubocki RJ Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements.J Mol Diagn. 2002; 4: 137-143Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar HR,8Sprouse JT Werling R Hanke D Lakey C McDonnel L Wood BL Sabath DE T-cell clonality determination using polymeras
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