Evaluation of T Cell Receptor Testing in Lymphoid Neoplasms
2001; Elsevier BV; Volume: 3; Issue: 4 Linguagem: Inglês
10.1016/s1525-1578(10)60664-2
ISSN1943-7811
AutoresDaniel A. Arber, Rita M. Braziel, Adam Bagg, Karen E. Bijwaard,
Tópico(s)T-cell and B-cell Immunology
ResumoTo evaluate current diagnostic methods used for the evaluation of T cell receptor (TCR) gene rearrangements, 24 different laboratories analyzed 29 lymphoid neoplasm samples of extracted DNA and paraffin-embedded tissue and were asked to complete a technical questionnaire related to the testing. Participating laboratories performed Southern blot and polymerase chain reaction (PCR) testing for rearrangements of the TCRβ chain gene and PCR for the TCRγ chain gene rearrangements. Of 14 laboratories performing TCRβ Southern blot analysis, there was complete agreement in 10 of 14 cases, with some false negative results obtained in 4 cases. No false positive results were obtained by Southern blot analysis. TCRβ PCR analysis was only performed by two laboratories, and only 47.1% of positive samples were detected. Twenty-one laboratory results were obtained for TCRγ PCR. This method showed an overall detection rate of 77.9% for T cell gene rearrangements with a 4.1% false positive rate, as compared to both TCRγ Southern blot analysis results and immunophenotyping. The detection rate for TCRγ PCR, however, significantly differed when extracted DNA samples from frozen tissue were compared to paraffin-embedded tissue (85.4% versus 65.9%;P = 0.0005). Significant differences in true positive results were obtained when laboratories using primers directed against multiple TCRγ variable regions (V1–8 plus one to three other primer sets) were compared to laboratories that used only a single set of TCR primers directed against the V1–8 (P < 0.0001). Other technical factors significantly affecting results were also identified. These findings provide useful data on the current state of diagnostic TCR testing, highlight the risk of false negative results for TCR testing directed against only portions of the TCRγgene, and identify limitations of testing of paraffin-embedded tissues in some laboratories. To evaluate current diagnostic methods used for the evaluation of T cell receptor (TCR) gene rearrangements, 24 different laboratories analyzed 29 lymphoid neoplasm samples of extracted DNA and paraffin-embedded tissue and were asked to complete a technical questionnaire related to the testing. Participating laboratories performed Southern blot and polymerase chain reaction (PCR) testing for rearrangements of the TCRβ chain gene and PCR for the TCRγ chain gene rearrangements. Of 14 laboratories performing TCRβ Southern blot analysis, there was complete agreement in 10 of 14 cases, with some false negative results obtained in 4 cases. No false positive results were obtained by Southern blot analysis. TCRβ PCR analysis was only performed by two laboratories, and only 47.1% of positive samples were detected. Twenty-one laboratory results were obtained for TCRγ PCR. This method showed an overall detection rate of 77.9% for T cell gene rearrangements with a 4.1% false positive rate, as compared to both TCRγ Southern blot analysis results and immunophenotyping. The detection rate for TCRγ PCR, however, significantly differed when extracted DNA samples from frozen tissue were compared to paraffin-embedded tissue (85.4% versus 65.9%;P = 0.0005). Significant differences in true positive results were obtained when laboratories using primers directed against multiple TCRγ variable regions (V1–8 plus one to three other primer sets) were compared to laboratories that used only a single set of TCR primers directed against the V1–8 (P < 0.0001). Other technical factors significantly affecting results were also identified. These findings provide useful data on the current state of diagnostic TCR testing, highlight the risk of false negative results for TCR testing directed against only portions of the TCRγgene, and identify limitations of testing of paraffin-embedded tissues in some laboratories. Lymphoid neoplasms are usually evaluated by a combination of methods that include hematoxylin and eosin stained tissue section morphology, often supplemented by immunophenotyping studies and possibly molecular genetic evaluation.1Harris NL Jaffe ES Diebold J Flandrin G Müller-Hermelink HK Vardiman J Lister TA Bloomfield CD The World Health Organization Classification of neoplastic diseases of the hematopoietic and lymphoid tissues: report of the clinical advisory committee meeting-Airlie House, Virginia, November 1997.J Clin Oncol. 1999; 17: 3835-3849Crossref PubMed Scopus (2523) Google Scholar, 2Arber DA Molecular diagnostic approach to non-Hodgkin's lymphoma.J Mol Diagn. 2000; 2: 178-190Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar The detection of a monoclonal cell population is usually helpful when a suspicion of malignancy exists and immunophenotyping methods for the detection of immunoglobulin light chain restriction are usually adequate for mature B cell neoplasms. No such simple immunophenotypic markers for clonality exist for T cell neoplasms, and molecular genetic testing for clonal rearrangements of T cell receptor genes are often useful in the evaluation of specimens suspicious for T cell lymphoma or leukemia. The four T cell receptor genes undergo variable-diversity-joining (VDJ) region or variable-joining (VJ) region rearrangements as part of normal T cell development, similar to the immunoglobulin heavy chain and light chain gene rearrangements of B cell development.3de Villartay J-P Hockett RD Coran D Korsmeyer SJ Cohen DI Deletion of the human T-cell receptor δ-gene by a site-specific recombination.Nature. 1988; 335: 170-174Crossref PubMed Scopus (144) Google Scholar, 4de Villartay J-P Pullman AB Andrade R Tzchachler E Colamenici O Neckers L Cohen DI Cossman J γ/δ lineage relationship within a consecutive series of human precursor T-cell neoplasms.Blood. 1989; 74: 2508-2518PubMed Google Scholar The T cell receptor δ locus (TCRδ) at chromosome region 14q11 is the first to rearrange, followed by TCRγ at 7q15,TCRβ at 7q34, and, finally, TCRα at 14q11. Approximately 95% of circulating T cells undergo all four rearrangements, but a small percentage of cells (γ/δ T cells) only undergo rearrangement of the first two genes. In the past, the most commonly used methods of detecting T cell receptor gene rearrangements used Southern blot analysis directed against the constant or joining regions of the TCRβ gene.5Cossman J Uppenkamp M Sundeen J Coupland R Raffeld M Molecular genetics and the diagnosis of lymphoma.Arch Pathol Lab Med. 1988; 112: 117-127PubMed Google Scholar Southern blot methods, however, are time-consuming, labor intensive, require a relatively large amount of fresh or frozen tissue, and require at least 5 to 10% clonal cells in the sample for detection. More recently, polymerase chain reaction (PCR)-based methods of detection of gene rearrangements have been used.2Arber DA Molecular diagnostic approach to non-Hodgkin's lymphoma.J Mol Diagn. 2000; 2: 178-190Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar These methods target rearrangements of either the TCRβ gene or the TCRγ gene; but because the TCRγ variable region is less complex than the TCRβ variable region, many laboratories prefer TCRγ testing. These methods are more rapid, require smaller amounts of tissue, may be used in paraffin-embedded tissues and can detect a smaller percentage of clonal cells than Southern blot analysis. To evaluate the utility and methodology used in laboratories performing T cell receptor clonality studies, the authors circulated a total of 29 samples of paraffin-embedded tissue and DNA extracted from frozen tissue from B and T cell lymphomas or leukemias to 21 diagnostic laboratories for PCR testing. Thirteen laboratories also performed Southern blot analysis on 14 of the samples, resulting in a total of 24 participating laboratories. The results of this sample exchange provide more information about the different testing methodologies used in diagnostic laboratories and their use in fresh/frozen and paraffin-embedded samples. Members of the Association for Molecular Pathology were surveyed for interest in participating in the sample exchange. Thirty respondents were sent samples and questionnaires, and responses were received from 24 laboratories. Participants were generally diagnostic laboratories and they were asked to perform their routine diagnostic assays. Frozen cells and available corresponding paraffin-embedded tissue of archived lymphoma and leukemia samples originally diagnosed at the Oregon Health Sciences University Department of Pathology were retrieved for use in this study. Lineage was assigned based on prior immunophenotyping studies in conjunction with morphological evaluation. Cases were classified according to the Revised European-American Classification of lymphoid neoplasms and the proposed World Health Organization Classification.1Harris NL Jaffe ES Diebold J Flandrin G Müller-Hermelink HK Vardiman J Lister TA Bloomfield CD The World Health Organization Classification of neoplastic diseases of the hematopoietic and lymphoid tissues: report of the clinical advisory committee meeting-Airlie House, Virginia, November 1997.J Clin Oncol. 1999; 17: 3835-3849Crossref PubMed Scopus (2523) Google Scholar, 6Harris NL Jaffe ES Stein H Banks PM Chan JK Cleary ML Delsol G De Wolf-Peeters C Falini B Gatter KC A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group.Blood. 1994; 84: 1361-1392PubMed Google Scholar Extracted DNA from 16 lymphoma/leukemia samples (9 B-lineage: 7 follicular lymphoma, 2 chronic lymphocytic leukemia/small lymphocytic lymphoma; 7 T-lineage: 4 peripheral T cell lymphoma, 1 mycosis fungoides/Sézary syndrome, 2 lymphoblastic lymphoma) was aliquoted and distributed to each participating laboratory. For DNA extraction, frozen cells were thawed, pelleted, and resuspended in an equal volume of lysis buffer, consisting of 50M Tris-HCL, pH 8.0, 100 mmol/L EDTA, supplemented with 0.9 mg/ml proteinase K and 0.5% SDS. The lysis mix was incubated for 16 hours at 55°C and extracted twice with an equal volume of phenol and once with chloroform/isoamylalcohol at a 24:1 ratio. DNA was precipitated with 0.1 volume of 7.5 mmol/L NH4OAc and 2.5 volumes of 100% ethanol, washed in 70% ethanol, and air-dried. The pellet was resuspended in TE (10 mmol/L Tris-HCl, pH 8.0, 1 mmol/L EDTA) and incubated for 1 hour at 55°C. If DNA was not completely dissolved, the TE incubation process was repeated as necessary. DNA concentration was determined and dilutions were made with TE to a final concentration of 0.1 μg/μl. 500 μl (50 μg DNA), or 100 μl (10 μg DNA) were aliquoted for distribution to the participating laboratories. Samples from 13 formalin-fixed paraffin-embedded lymphoma/leukemia specimens were also distributed. These included 11 paraffin samples that corresponded to frozen cell specimens (5 B-lineage: 4 follicular lymphoma and 1 chronic lymphocytic leukemia/small lymphocytic lymphoma; 6 T-lineage: 4 peripheral T cell lymphoma, 1 lymphoblastic lymphoma and 1 mycosis fungoides, Sézary syndrome), and two additional paraffin-embedded specimens (1 B-lineage: mantle cell lymphoma; 1 T-lineage: peripheral T cell lymphoma). For paraffin-embedded tissues, four or five 10-μm sections of each block were distributed in tubes to each participating laboratory and DNA extraction was performed in the individual laboratories. Laboratories were not aware that that some frozen and paraffin samples were from the same tumor. Results were compared between laboratories with the exception of Southern blot analysis for rearrangements of the T cell receptor γ chain gene (TCRγ), which was performed in one laboratory for comparison to the polymerase chain reaction testing for rearrangements of this locus. For TCRγ Southern blot, 16 μg of extracted DNA was digested with HindIII and EcoRI for 3 hours. The digested DNA was loaded on a 0.7% agarose gel and run at 34 V for 16 hours at room temperature. After electrophoresis, the DNA was transferred overnight with 0.4 N NaOH onto a nylon membrane. The blots were hybridized with H60 probe (ATCC 59585, Rockville, MD).7Lefranc MP Rabbitts TH Two tandemly organized human genes encoding the T-cell gamma constant-region sequences show multiple rearrangement in different T-cell types.Nature. 1985; 316: 464-466Crossref PubMed Scopus (295) Google Scholar H60 probe was labeled with P-32 nucleotides by use of a random primer labeling kit (Amersham Pharmacia, Arlington Heights, IL). The blots were washed with 0.1X SSC and 0.1% SDS, and exposed to X-ray films for 5 days. The presence of additional bands or loss of bands when compared to a germline control was considered evidence of a TCRγ gene rearrangement. The submitting laboratory, using their own established guidelines, interpreted the test results. Specific guidelines for interpretation were not proposed by the exchange. Samples were accompanied by a technical questionnaire related to the individual test performed and laboratories were requested to return the completed questionnaire, a summary of results and copies of diagnostic radiographs and/or gels used for interpreting results. χ2 analysis of probability was performed using GB-STAT version 7.0 (Dynamic Microsystems, Inc., Silver Spring, MD). Probability (P) values of 0.05 or less were considered statistically significant. Results were obtained from 24 laboratories, including one laboratory that performed TCRγ PCR by two different methods and was counted twice to give a total of 25 laboratory results. Thirteen laboratories performed Southern blot analysis for TCR gene rearrangements on some or all of the extracted DNA specimens, two performed TCRβ PCR, and 21 performed TCRγPCR. The technical questionnaire for TCR Southern blot analysis indicated that laboratories routinely used two to five different restriction enzymes with the majority of laboratories(9 of 13) using BamHI, EcoRI, and HindIII. Of the 11 laboratories responding, three required only one rearranged band for a positive result and the remaining eight required two rearranged bands before interpreting a case as rearranged. Seven laboratories used isotopic probes labeled in their own laboratory and seven used non-isotopic methods. Six laboratories used JβI-II probes only, four used CTβ probes only and three used both. The technical questionnaire on TCRγ PCR indicated that paraffin section extraction methods varied from organic in 7 (33%), inorganic in 7 (33%), and crude lysate in 5 (24%) laboratories. No information was received from two (10%) of the participating laboratories. Twelve centers (57%) used a “hot start” PCR approach and 8 (38%) used standard PCR. One center gave no information regarding the PCR approach. All 20 responding laboratories used a non-nested PCR method, but the TCRγ variable regions covered by the primers varied. Nine laboratories (43%) used a combination of primers directed against all 11 Vγ regions (Vγ 1–8, 9, 10, 11) as well as a multiplex of Jγ primers (Group 1);8Trainor KJ Brisco MJ Wan JH Neoh S Grist S Morley AA Gene rearrangement in B- and T-lymphoproliferative disease detected by the polymerase chain reaction.Blood. 1991; 78: 192-196PubMed Google Scholar, 9Algara P Soria C Martinez P Sanchez L Villuendas R Garcia P Lopez C Orradre JL Piris MA Value of PCR detection of TCRγ gene rearrangement in the diagnosis of cutaneous lymphocytic infiltrates.Diagn Mol Pathol. 1994; 3: 275-282Crossref PubMed Scopus (41) Google Scholar, 10Greiner TC Raffeld M Lutz C Dick F Jaffe ES Analysis of T cell receptor-γ gene rearrangements by denaturing gradient gel electrophoresis of GC-clamped polymerase chain reaction products: correlation with tumor-specific sequences.Am J Pathol. 1995; 146: 46-55PubMed Google Scholar, 11Theodorou J Bigorgne C Delfau M-H Lahet C Cochet G Vidaud M Raphael M Gaulard P Farcet J-P VJ rearrangements of the TCRγ 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 4 centers (19%) used a Jγ multiplex with primers directed against Vγ 1–8, 10 and 11 (Group 2);12McCarthy KP Sloane JP Kabarowski JHS Wiedemann LM The rapid detection of clonal T-cell proliferations in patients with lymphoid disorders.Am J Pathol. 1991; 138: 821-828PubMed Google Scholar, 13McCarthy KP Sloane JP Kabarowski JHS Matutes E Wiedemann LM A simplified method of detection of clonal rearrangements of the T-cell receptor-γ chain gene.Diagn Mol Pathol. 1992; 1: 173-179PubMed Google Scholar, 14Slack DN McCarthy KP Wiedmann LM Sloane JP Evaluation of sensitivity, specificity, and reproducibility of an optimized method for detecting clonal rearrangements of immunoglobulin and T-cell receptor genes in formalin-fixed, paraffin-embedded sections.Diagn Mol Pathol. 1993; 2: 223-232Crossref PubMed Scopus (122) Google Scholar, 15Diss TC Watts M Pan LX Burke M Linch D Isaacson PG The polymerase chain reaction in the demonstration of monoclonality in T-cell lymphomas.J Clin Pathol. 1995; 48: 1045-1050Crossref PubMed Scopus (132) Google Scholar 1 laboratory (5%) used primers directed against Vγ 1–8, and 9 with a multiplex of Jγ primers (Group 3);8Trainor KJ Brisco MJ Wan JH Neoh S Grist S Morley AA Gene rearrangement in B- and T-lymphoproliferative disease detected by the polymerase chain reaction.Blood. 1991; 78: 192-196PubMed Google Scholar and, 5 (25%) used a single set of primers directed against Vγ 1–8 and Jγ (Group 4).16Veelken H Tycko B Sklar J Sensitive detection of clonal antigen receptor gene rearrangements for the diagnosis and monitoring of lymphoid neoplasms by a polymerase chain reaction-mediated ribonuclease protection assay.Blood. 1991; 78: 1318-1326PubMed Google Scholar, 17Benhattar J Delacretaz F Martin P Chaubert P Improved polymerase chain reaction detection of clonal T-cell lymphoid neoplasms.Diagn Mol Pathol. 1995; 4: 108-112Crossref PubMed Scopus (89) Google Scholar, 18Kaul K Petrick M Herz B Cheng TC Detection of clonal rearrangement of the T-cell receptor gamma gene by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP).Mol Diagn. 1996; 1: 131-137Crossref PubMed Scopus (10) Google Scholar, 19Guitart J Kaul K A new polymerase chain reaction-based method for the detection of T-cell clonality in patients with possible cutaneous T-cell lymphoma.Arch Dermatol. 1999; 135: 158-162Crossref PubMed Scopus (37) Google Scholar The Vγ regions covered by the PCR primers were not available for 2 laboratories. The Jγ primers used were variable within the groups. In Group 1, two laboratories used a multiplex of J1, JP, JP1 and JP2; 1 laboratory each used the following combinations: J2, JP, JP1 and JP2; J1 and J2; J2 and JP2; J1/2 and JP; J1/2, JP, JP1 and JP2; and J1/2 and JP1. Two laboratories in Group 2 used multiplex of J2 and JP2, while the other 2 laboratories in this group used the combinations of J2, JP and JP2, and J2, J1/2 and JP2. The single laboratory in Group 3 reported use of a multiplex of J2, JP, and JP1. Three of the laboratories in Group 4 reported use of a single set of J2 primers, one used a single set of J1/2 primers and specifics of the single primer set used were not provided by one laboratory. PCR products were analyzed on polyacrylamide gel in 14 laboratories (66%), by capillary electrophoresis in 3 (14%) and by agarose, MetaPhor (Cambrex, East Rutherford, NJ), and denaturing gradient gel electrophoresis (DGGE) in 1 laboratory each. The method of analysis was not reported for one laboratory. Denaturing conditions (heteroduplex) were used in 6 of the 20 laboratories with data. Ethidium bromide staining was used by 12 of 19 reporting labs, by silver staining in 2 labs, by SYBR green staining in 2 laboratories, and by Gelstar, incorporated radionucleotide and chemiluminescence in 1 laboratory each. Types of controls included patient samples (9 laboratories; 43%) and cell lines (10 laboratories; 47%). Types of controls were not reported for 2 laboratories (10%). Fifteen laboratories gave information on the type of sensitivity controls that they used, with diluted cells used by 3 and diluted DNA by 12. The range of sensitivities predicted by the laboratories was 0.001 to 10% for frozen samples and 0.01 to 10% for paraffin samples. Laboratories predicted that their TCRγmethodology could detect 75 to 95% of clonal T cell rearrangements with a mean of 86%. Southern blot analysis showed rearrangement of the TCRγ gene in all cases of T cell lymphoma and none of the B cell neoplasms. The Southern blot results are summarized in Table 1. Although all laboratories that performed Southern blot analysis for TCR gene rearrangements did not test every specimen, presumably due to a lack of sufficient DNA for all tests covered by the sample exchange, there was concordance among all testing laboratories on 10 of 14 samples. The complete concordance was on four B cell neoplasms (all TCRβ germline) and six T cell neoplasms (all TCRβ rearranged). Differences in results in four cases included one follicular lymphoma (eight laboratories TCRβ rearranged and two germline), one mycosis fungoides/Sézary syndrome (11 laboratories TCRβ rearranged, one germline), and two peripheral T cell lymphomas (one case with 4 laboratories TCRβ rearranged and three germline; one case with 12 laboratories TCRβ rearranged and one germline). For the 5 laboratories with a discordant result, no trend related to probe type was identified when compared to the overall results. Two used CTβ probes and three used JβI-II. Two of the laboratories with discordant results used isotope-labeled probes and 3 used non-isotopic probes. No discordant results were reported from the 3 laboratories that used both CTβ and JβI-IIprobes, although 1 of those laboratories only tested 3 of the 14 available samples. Based on the consensus of the participating laboratories of no TCRβ rearrangements detected 6 of 7 of the B cell neoplasms, and apparent dual T and B cell rearrangements in one B cell lymphoma, the results of the survey were interpreted as representing no false positive results for TCRβ Southern blot analysis.Table 1Summary of Southern Blot ResultsSample no.Lineage and diagnosisJHTCRγTCRβ1B-chronic lymphocytic leukemiaRG10/10 (100%)G2B-chronic lymphocytic leukemiaRG10/10 (100%)G3T-lymphoblastic lymphomaGR12/12 (100%)R4B-follicular lymphoma, grade IRG8/10 (80%)R2/10 (20%)G5T-lymphoblastic lymphomaGR11/11 (100%)R6T-mycosis fungoides/Sézary syndromeGR11/12 (92%)R1/12 (8%)G7T-peripheral T cell lymphomaGR4/7 (57%)R3/7 (43%)G8B-follicular lymphoma, grade IIIRG10/10 (100%)G9T-peripheral T cell lymphomaGR12/13 (92%)R1/13 (8%)G10B-follicular lymphoma, grade IRG10/10 (100%)G11T-peripheral T cell lymphomaGR7/7 (100%)R12B-follicular lymphoma, grade IRG9/9 (100%)G13B-follicular lymphoma, grade IRG10/10 (100%)G14T-peripheral T cell lymphomaGR12/12 (100%)RR, rearranged; G, germline. Open table in a new tab R, rearranged; G, germline. Polymerase chain reaction testing for TCRβ was only performed in two laboratories, limiting the evaluation of this assay as a diagnostic test. Table 2 summarizes the results from these laboratories, compared to the consensus Southern blot results for TCRβ (including one TCRβ-positive B cell neoplasm). Only 47.1% of expected TCRβ rearrangements were detected by this method with a 15% false positive rate.Table 2TCR PCR ResultsSample no.Lineage and diagnosisSpecimen typeTCRβ SBTCRβ PCRTCRγ SBTCRγ PCR1B-chronic lymphocytic leukemiaFrozenG1/2G6/18 (33%)2aB-chronic lymphocytic leukemiaFrozenG0/2G0/19 (0%)2bB-chronic lymphocytic leukemiaParaffinGNDG1/15 (7%)3T-lymphoblastic lymphomaFrozenR2/2R21/21 (100%)4B-follicular lymphoma, grade IFrozenR0/2G0/19 (0%)5aT-lymphoblastic lymphomaFrozenR1/2R21/21 (100%)5bT-lymphoblastic lymphomaParaffinRNDR5/9 (56%)6aT-mycosis fungoides/Sézary syndromeFrozenR1/2R9/20 (45%)6bT-mycosis fungoides/Sézary syndromeParaffinRNDR1/8 (13%)7aT-peripheral T cell lymphomaFrozenR2/2R20/20 (100%)7bT-peripheral T cell lymphomaParaffinR0/1R14/17 (82%)8aB-follicular lymphoma, grade IIIFrozenG0/2G0/19 (0%)8bB-follicular lymphoma, grade IIIParaffinGNDG0/6 (0%)9aT-peripheral T cell lymphomaFrozenR0/2R14/21 (67%)9bT-peripheral T cell lymphomaParaffinRNDR2/8 (25%)10aB-follicular lymphoma, grade IFrozenG0/2G0/21 (0%)10bB-follicular lymphoma, grade IParaffinGNDG0/10 (0%)11aT-peripheral T cell lymphomaFrozenR1/2R18/20 (90%)11bT-peripheral T cell lymphomaParaffinR0/1R12/16 (75%)12aB-follicular lymphoma, grade IFrozenG0/1G0/19 (0%)12bB-follicular lymphoma, grade IParaffinG0/1G1/15 (7%)13aB-follicular lymphoma, grade IFrozenG0/2G0/20 (0%)13bB-follicular lymphoma, grade IParaffinGNDG0/13 (0%)14aT-peripheral T cell lymphomaFrozenR0/2R20/21 (95%)14bT-peripheral T cell lymphomaParaffinRNDR12/17 (71%)15T-peripheral T cell lymphomaParaffinNA1/1NA2/16 (13%)16B-mantle cell lymphomaParaffinNA1/2NA2/15 (13%)17B-follicular lymphoma, grade IFrozenNA1/2NA0/17 (0%)18B-follicular lymphoma, grade IIIFrozenNA0/2NA0/16 (0%)R, rearranged; G, germline; Frozen, extracted DNA from previously frozen samples; ND, not tested; NA, material not submitted for Southern blot testing. Open table in a new tab R, rearranged; G, germline; Frozen, extracted DNA from previously frozen samples; ND, not tested; NA, material not submitted for Southern blot testing. Polymerase chain reaction testing for TCRγ was performed in 21 laboratories, and the results are summarized in Table 2, Table 3. Overall, these tests detected 77.9% of expected T cell gene rearrangements. There was a significant decrease in the ability to detect the gene rearrangement in paraffin-embedded tissue, ranging from 85.4% of extracted DNA samples and 65.9% of paraffin samples (P = 0.0005). A 4.1% false positive rate was detected, overall, and no difference was seen between paraffin and extracted DNA samples. Almost 22% of samples, however, were not tested, presumably due to the lack of amplification of an internal control gene. A lack of testing was significantly higher for the paraffin embedded samples (7.1% of extracted DNA samples versus 39.6% for paraffin samples; P < 0.0001).Table 3Combined Results of All Laboratories for TCR PCRFrozen TPFrozen FPFrozen not testedParaffin TPParaffin FPParaffin not testedTotal TPTotal FPTotal not testedTCRβ7/142/171/321/31/320/268/173/2021/5850.0%11.8%3.1%33.3%33.3%76.9%47.1%15.0%36.2%TCRγ123/1446/16824/33660/914/74108/273183/23510/242132/60985.4%3.6%7.1%65.9%5.4%39.6%77.9%4.1%21.7%A comparison of extracted DNA samples (frozen) to paraffin samples showed a significant increase in true positive results for frozen testing (P = 0.0005), and a significant increase in cases not tested for paraffin samples (P < 0.0001). No difference in false positive results was observed. TP, true positive; FP, false positive. Open table in a new tab A comparison of extracted DNA samples (frozen) to paraffin samples showed a significant increase in true positive results for frozen testing (P = 0.0005), and a significant increase in cases not tested for paraffin samples (P < 0.0001). No difference in false positive results was observed. TP, true positive; FP, false positive. The TCRγ results were further analyzed by paraffin extraction methods, PCR method (hot start versus standard), DNA analysis method (capillary electrophoresis versuspolyacrylamide gel and denaturing versus non-denaturing gel analysis) and TCRγ variable and joining regions covered by the PCR primers. These results are summarized in Table 4, Table 5, Table 6, Table 7, Table 8. The use of inorganic extraction methods of paraffin tissue resulted in a slight, but significant (P = 0.0059) increase in false positive results, when compared to organic and crude lysate methods (Table 4). Although a significant difference in results was not observed when hot start PCR was compared to standard PCR, more cases could not be analyzed due to lack of amplifiable DNA when standard PCR methods were used (P = 0.0053) (Table 5). When capillary electrophoresis was compared to polyacrylamide gel analysis of DNA products, a slight increase in paraffin false positive results was observed with capillary electrophoresis (2/12 or 16.7% versus 1/53 or 1.9%; P = 0.0276) (Table 6). However, significantly fewer cases could be analyzed using polyacrylamide gels (20.2% polyacrylamide gel paraffin cases not tested versus 10.3% for capillary electrophoresis; P = 0.0316). Although there was a slight increase in extracted frozen samples not tested when non-denaturing conditions were compared to denaturing gels (P = 0.0257), no other significant differences between these methods were identified (Table 7).Table 4TCRγ Paraffin Extraction MethodsExtraction methodLab nTPFPNot testedOrganic721/34 (61.8%)0/30 (0%)27/91 (29.7%)Inorganic726/34 (76.5%)4/23 (17.4%)34/91 (37.4%)Crude lysate611/26 (42.3%)0/26 (0%)26/78 (33.3%)No data23/4 (75.0%)0/1 (0%)21/26 (80.8%)Contingency table analysis revealed a significant difference (P = 0.0059) when the three methods were compared for false positive results. TP, true positive; FP, false positive. Open table in a new tab Table 5TCRγ Hot Start versus Standard PCRLab nFrozen TPFrozen FPFrozen not testedParaffin TPParaffin FPParaffin not testedTotal TPTotal FPTotal not testedStandard847 /564 /5814 /12820 /311 /2746 /10467 /876 /8560 /23283.9%6.9%10.9%64.5%3.7%44.2%77.0%7.1%25.9%Hot start1272 /832 /1063 /19237 /563 /4654 /156109 /135 /15257 /34886.7%1.9%1.6%66.1%6.5%34.6%93.3%16.4%78.4%No data14 /50 /47 /163 /40 /18 /137 /90 /515 /2980.0%0%43.8%75.0%0%61.5%77.8%0%51.7%The difference between frozen samples not tested and total samples not tested using standard and hot start PCR methods was statistically significant (P = 0.0002 and P =
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