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• W2125202362 abstract "Molecular markers for minimal residual disease in B-lineage acute lymphoblastic leukemia were identified by determining, at the time of diagnosis, the repertoire of rearrangements of the immunoglobulin heavy chain (IGH) gene using segment-specific variable (V), diversity (D), and junctional (J) primers in two different studies that involved a total study population of 75 children and 18 adults. This strategy, termed repertoire analysis, was compared with the conventional strategy of identifying markers using family-specific V, D, and J primers for a variety of antigen receptor genes. Repertoire analysis detected significantly more markers for the major leukemic clone than did the conventional strategy, and one or more IgH rearrangements that were suitable for monitoring the major clone were detected in 96% of children and 94% of adults. Repertoire analysis also detected significantly more IGH markers for minor clones. Some minor clones were quite large and a proportion of them would not be able to be detected by a minimal residual disease test directed to the marker for the major clone. IGH repertoire analysis at diagnosis has potential advantages for the identification of molecular markers for the quantification of minimal residual disease in acute lymphoblastic leukemia cases. An IGH marker enables very sensitive quantification of the major leukemic clone, and the detection of minor clones may enable early identification of additional patients who are prone to relapse. Molecular markers for minimal residual disease in B-lineage acute lymphoblastic leukemia were identified by determining, at the time of diagnosis, the repertoire of rearrangements of the immunoglobulin heavy chain (IGH) gene using segment-specific variable (V), diversity (D), and junctional (J) primers in two different studies that involved a total study population of 75 children and 18 adults. This strategy, termed repertoire analysis, was compared with the conventional strategy of identifying markers using family-specific V, D, and J primers for a variety of antigen receptor genes. Repertoire analysis detected significantly more markers for the major leukemic clone than did the conventional strategy, and one or more IgH rearrangements that were suitable for monitoring the major clone were detected in 96% of children and 94% of adults. Repertoire analysis also detected significantly more IGH markers for minor clones. Some minor clones were quite large and a proportion of them would not be able to be detected by a minimal residual disease test directed to the marker for the major clone. IGH repertoire analysis at diagnosis has potential advantages for the identification of molecular markers for the quantification of minimal residual disease in acute lymphoblastic leukemia cases. An IGH marker enables very sensitive quantification of the major leukemic clone, and the detection of minor clones may enable early identification of additional patients who are prone to relapse. The magnitude of the early response to chemotherapy is a powerful prognostic factor in acute lymphoblastic leukemia (ALL)1Brisco MJ Condon J Hughes E Neoh SH Sykes PJ Seshadri R Toogood I Waters K Tauro G Ekert H Outcome prediction in childhood acute lymphoblastic leukaemia by molecular quantification of residual disease at the end of induction.Lancet. 1994; 343: 196-200Abstract PubMed Google Scholar2Cave H van der Werff ten Bosch J Suciu S Guidal C Waterkeyn C Otten J Bakkus M Thielemans K Grandchamp B Vilmer E Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. European Organization for Research and Treatment of Cancer–Childhood Leukemia Cooperative Group.N Engl J Med. 1998; 339: 591-598Crossref PubMed Scopus (638) Google Scholar3van Dongen JJ Seriu T Panzer-Grumayer ER Biondi A Pongers-Willemse MJ Corral L Stolz F Schrappe M Masera G Kamps WA Gadner H van Wering ER Ludwig WD Basso G de Bruijn MA Cazzaniga G Hettinger K van der Does-van den Berg A Hop WC Riehm H Bartram CR Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood.Lancet. 1998; 352: 1731-1738Abstract Full Text Full Text PDF PubMed Scopus (818) Google Scholar and, to assess this, rearrangements of the immunoglobulin and/or T-cell receptor genes are now widely used as molecular markers for measuring the level of minimal residual disease (MRD). The most common approach for identifying markers, used by many laboratories including the Sydney group, involves screening for a variety of immunoglobulin and T-cell receptor gene rearrangements by polymerase chain reaction (PCR) using family-specific primers for variable (V), diversity (D), and junctional (J) segments.4Foroni L Hoffbrand AV Molecular analysis of minimal residual disease in adult acute lymphoblastic leukaemia.Bailliere's Best Pract Clin Haematol. 2002; 15: 71-90Abstract Full Text PDF PubMed Scopus (21) Google Scholar5Neale GA Coustan-Smith E Stow P Pan Q Chen X Pui CH Campana D Comparative analysis of flow cytometry and polymerase chain reaction for the detection of minimal residual disease in childhood acute lymphoblastic leukemia.Leukemia. 2004; 18: 934-938Crossref PubMed Scopus (141) Google Scholar6Pongers-Willemse MJ Seriu T Stolz F d'Aniello E Gameiro P Pisa P Gonzalez M Bartram CR Panzer-Grumayer ER Biondi A San Miguel JF van Dongen JJ Primers and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets: report of the BIOMED-1 CONCERTED ACTION: investigation of minimal residual disease in acute leukemia.Leukemia. 1999; 13: 110-118Crossref PubMed Scopus (325) Google Scholar7van 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: 2257-2317Crossref PubMed Scopus (2518) Google Scholar8Zhou J Goldwasser MA Li A Dahlberg SE Neuberg D Wang H Dalton V McBride KD Sallan SE Silverman LB Gribben JG Dana-Farber Cancer Institute ALLC Quantitative analysis of minimal residual disease predicts relapse in children with B-lineage acute lymphoblastic leukemia in DFCI ALL Consortium Protocol 95-01.Blood. 2007; 110: 1607-1611Crossref PubMed Scopus (121) Google Scholar The Flinders group has developed a somewhat different approach, termed repertoire analysis, which uses primers directed against individual V, D, and J segments of the immunoglobulin heavy chain gene (IGH) to identify rearrangements present at diagnosis in B-lineage ALL. An initial study by the Flinders group showed that the repertoire strategy had some advantages and consequently a second study was performed in collaboration with the Sydney group to enable a direct comparison of the two approaches using the same set of patient samples. In this paper we report the results of both sets of studies, which illustrate the utility of repertoire analysis for marker detection and highlight the occurrence of clonal evolution in ALL. The first study used bone marrow samples obtained at diagnosis from 25 children and 18 adults with B-lineage ALL. Ethical approval was obtained for the procurement of all samples. Genomic DNA was extracted using the Qiagen Flexigene DNA kit according to the manufacturer's instructions. The second study used bone marrow samples obtained from another 50 children with B-lineage ALL. The patients came from a series of 50 consecutive patients with B-lineage ALL treated at Sydney Children's Hospital in the Australian and New Zealand Children's Hematology Oncology Group (ANZCHOG) Study 8 clinical trial. DNA was extracted from Ficoll purified mononuclear cells using Machery-Nagel Nucleobond column purification according to the manufacturer's instructions. The detailed methods below are those used for repertoire analysis in the Flinders laboratory. The methods used for conventional analysis in the Sydney laboratory are described subsequently. Unless otherwise stated, duplicate amplifications were performed, each in a volume of 25 μl and containing 2 mmol/L Tris-HCl (pH 8.4), 50 mmol/L KCl, 4 mmol/L MgCl2, 200 μmol/L each of dATP, dUTP, dCTP, and dGTP, 100 ng of each primer, 1 unit of Platinum Taq (Invitrogen) and, for Q-PCR, 4 pmol of a TaqMan probe to a conserved sequence in the J segment. Cycling conditions were 92°C for 15 seconds followed by 58°C for 1 minute and 72°C for 15 seconds. Unless otherwise stated, the mass of genomic DNA in each PCR was 2 ng in the first study and 20 ng in the second study. The sequences of all primers and probes used for IGH repertoire analysis are shown in Table 1. The primers were designed to cover as comprehensively as possible the 52 functional VH regions listed in the IMGT database.9Lefranc MP Giudicelli V Kaas Q Duprat E Jabado-Michaloud J Scaviner D Ginestoux C Clement O Chaume D Lefranc G IMGT, the international ImMunoGeneTics information system.Nucleic Acids Res. 2005; 33: D593-D597Crossref PubMed Scopus (255) Google Scholar Thus, 32 primers were designed to match just one VH segment; eight primers to match two VH segments each; and one primer to match four homologous VH4 segments. Two TaqMan probes were used, the J probe1 for J1 and J3–J6 and J probe2 for the J2 segment. It should be noted that the consensus primer FR2B amplifies Vh1 family segments relatively inefficiently.Table 1Primer Sequences Used for Repertoire AnalysisVH segment-specific primersV1.025′-ATCAACCCTAACAGTGGTGG-3′V3.335′-AGTGGGTGGCAGTTATATGG-3′V1.035′-GCTGGCAATGGTAACACAAAA-3′V3.435′-GGTCTCTCTTATTAGTTGGGA-3′V1.085′-ACCTAACAGTGGTAACACAGG-3′V3.495′-ATGGTGGGACAACAGAATACA-3′V1.185′-GGGATGGATCAGCGCTT-3′V3.535′-GTGGGTCTCAGTTATTTATAGC-3′V1.245′-TGGAGGTTTTGATCCTGAAGA-3′V3.645′-CTCAGCTATTAGTAGTAATGGG-3′V1.455′-ACACCTTTCAATGGTAACACC-3′V3.725′-AAACAAAGCTAACAGTTACACC-3′V1.465′-GGGAATAATCAACCCTAGTGG-3′V3.735′-AAGCAAAGCTAACAGTTACG-3′V1.585′-GATAGGATGGATCGTCGTTG-3′V3.745′-TCACGTATTAATAGTGATGGGA-3′V1.695′-TCATCCCTATCTTTGGTACAG-3′V3D5′-TCCATTAGTGGTGGTAGCA-3′V2.055′-ACTCATTTATTGGAATGATGATAAG-3′V4.045′-CCATCAGCAGTAGTAACTGG-3′V2.265′-ACACATTTTTTCGAATGACGAA-3′V4.285′-GCAGTAGTAACTGGTGGG-3′V2.705′-TGATTGGGATGATGATAAATTCT-3′V4.30.15′-GACTGGTGAAGCCTTCACA-3′V3.075′-AGCAAGATGGAAGTGAGAAA-3′V4.345′-ATGGTGGGTCCTTCAGTG-3′V3.095′-GGAATAGTGGTAGCATAGGC-3′V4.395′-AGAGTCGAGTCACCATATCC-3′V3.115′-CATTAGTAGTAGTGGTAGTACCAT-3′V4.595′-CTGGTGGCTCCATCAGTA-3′V3.135′-TCTCAGCTATTGGTACTGC-3′V4.615′-GTCTCTGGTGGCTCCG-3′V3.155′-GCGGTATTAAAAGCAAAACTG-3′V5.515′-CTGGTGACTCTGATACCAGA-3′V3.205′-GCTGGAGTGGGTCTCT-3′V5A5′-ATCCTAGTGACTCTTATACCAAC-3′V3.215′-CATCCATTAGTAGTAGTAGTAGTT-3′V6.015′-CATACTACAGGTCCAAGTGG-3′V3.235′-GTGGGTCTCAGCTATTAGTG-3′V7.4.15′-GATCAACACCAACACTGGG-3′V3.305′-AGTGGGTGGCAGTTATATCA-3′6 DH primers for D-J partial rearrangementsD1 out5′-ACCCAGGAGGCCCCAGAGCTCAGGG-3′D1 in5′-CCCGGTCGGATTCTGAACAGCCCCGA-3′D2 out5′-CACCMGKAGGGACAGGAGGATTTTGTGGG GG-3′D2 in5′-GGAGGATTTTGTGGGGGCTCGTGTCACTG-3′D3 out5′-GCCCTGGACATCCCGGGTTTCCCCAGG-3′D3 in5′-GGGTTTCCCCAGGCCTGGCGGTAGGTTT-3′D4 out5′-TGGACCAGGGCCTGCGTGGGAAAGG-3′D4 in5′-GCGTGGGAAAGGCCTCTGGSCACACTC-3′D5 out5′-GCCCCGCCTCCAGTTCCAGGTGTGG-3′D5 in5′-GCCTCCAGTTCCAGGTGTGGTTATTGTCA GG-3′D6 out5′-GNGGKGCTGAGCCCAGCAAGGGAAGG-3′D6 in5′-GCCCAGCAAGGGAAGGCCCCCAAACA-3′D7 out5′-CAGGCCCCCTACCAGCCGCAGGG-3′D7 in5′-AGCCGCAGGGTTTTGGCTGAGCTG-3′JH specific primers (from the intron between J segments)J1a5′-TCCCCAAGTCTGAAGCCA-3′J4a5′-TCCGGGGCTCTCTTGG-3′J1b5′-CGACCTCCTTTGCTGAG-3′J4b5′-TTGCCCCTCGTCTGTGT-3′J2a5′-GGAGGGGGCTGCAGTG-3′J5a5′-GCAAGCTGAGTCTCCCT-3′J2b5′-GGCTGCAGACCCCAGA-3′J5b5′-CTTTCTTTCCTGACCTCCAA-3′J3a5′-CCCAGCTCCAGGACAGA-3′J6a5′-ACAAAGGCCCTAGAGTGG-3′J3b5′-CAGCGCAGACCAAGGA-3′J6b5′-CCCACAGGCAGTAGCAG-3′Set of 7 VH primers for multiplexing for pre-amplificationIgH pre15′-GGCTCTGGCAGGTGCAGCTGGTGGAGTCTGG-3′IgH pre55′-GGCTCTGGCAGGTGCAGCTGGTGGAGTCCGG-3′IgH pre25′-GGCTCTGGCAGGTGCAGCTGGTGCAGTCTGG-3′IgH pre65′-GGCTCTGGCAGGTACAGCTGCAGCAGTCAGG-3′IgH pre35′-GGCTCTGGCAGGTCACCTTGAAGGAGTCTGG-3′IgH pre75′-GGCTCTGGCAGGTGCAGCTACAGCAGTGGGG-3′IgH pre45′-GGCTCTGGCAGGTGCAGCTGCAGGAGTCGGG-3′Consensus primersFR3A (in V)5′-ACACGGCCGTGTATTACTGT-3′VLJH (in J)5′-GTGACCAGGGTNCCTTGGCCCCAG-3′FR2B (in V)5′-GTCCTGCAGGCYYCCGGRAARRGTCTGGAGTGG-3′ELJH (in J)5′-TGAGGAGACGGTGACCAGGATCCCTTGGCCCCAG-3′TaqMan probes in JJ probe15′-TCACCGTCTCCTCAGG-3′J probe25′-TCACTGTCTCCTCAGG-3′ Open table in a new tab Preamplification performed two functions. It enabled study of a relatively large amount of genomic DNA to minimize sampling error and facilitate detection of low-abundance rearrangements, and it provided sufficient material for the large number of PCRs used in subsequent analysis. Samples from 10 children and 10 adults in the first study and the 50 patients in the collaborative study were preamplified using IGH generic primers. Two or three replicate samples, each of 50 ng of genomic DNA, were preamplified for 15 cycles in a multiplex PCR that included 20 ng each of seven V primers (IGH pre 1–7) designed to cover all framework 1 sequences in the germline VH regions and 20 ng each of six J primers (1a, 2a, 3b, 4a, 5a, 6a) designed to cover all J sequences of the IGH locus. Consensus forward primers FR3A and FR2B and/or the pool of seven DOUT segment primers were tested against primers (both Ja and Jb) for each of the six individual J segments by Q-PCR using 100 ng of each primer, 20 ng of genomic DNA, and both the J1 and J2 probes. The J segment involved in a rearrangement was identified from the cycle threshold (Ct) value together with visualization of an appropriately sized band after electrophoresis. V segments were identified after determining J segment usage. In the first study, V segment usage was determined using genomic DNA, although in 10 children and 10 adults it was also determined using preamplified DNA. In the second study, a 1:1000 dilution of preamplified DNA was used to identify candidate V segments, which were confirmed using 50 ng of genomic DNA. V segment identification involved separate PCR testing of 41 forward VH segment primers against the previously identified reverse J primer. Where a V-specific primer gave a Ct within 12 cycles of that given by the control primers, FR3A and FR2B, electrophoresis was used to confirm the presence of a band of appropriate size, in the range of 200 to 500 bp. In the first study, screening for DJ rearrangements was only performed if a complete VDJ rearrangement had not been identified; however, in the collaborative study, all samples were screened for DJ rearrangements. Incomplete D-J rearrangements were identified by Q-PCR on genomic DNA with a mixture of the seven forward DOUT segment primers tested with the six individual reverse J primers, followed by testing individual DIN primers with the previously identified J primer. Amplification of a DJ rearrangement gave a sharp gel band in the range of 90 to 300 bp. Candidate rearrangements amplified using specific V-J or D-J primer pairs were cut from gels and sequenced using an ABI 3100 genetic analyzer. To confirm the identity of V, D, and J regions used, sequences were screened using the IMGT database9Lefranc MP Giudicelli V Kaas Q Duprat E Jabado-Michaloud J Scaviner D Ginestoux C Clement O Chaume D Lefranc G IMGT, the international ImMunoGeneTics information system.Nucleic Acids Res. 2005; 33: D593-D597Crossref PubMed Scopus (255) Google Scholar and V-QUEST tool.10Giudicelli V Chaume D Lefranc MP IMGT/V-QUEST, an integrated software program for immunoglobulin and T cell receptor V-J and V-D-J rearrangement analysis.Nucleic Acids Res. 2004; 32: W435-W440Crossref PubMed Scopus (248) Google Scholar Rearrangements were assumed to belong to the same lineage if they appeared to have been created by V region replacement, ie, they shared the same J region, N-J junction and N region, and possibly part of the D region as well. In assigning the order in which clones arose, it was assumed that in V region replacement, a downstream V region is replaced by another V segment further upstream. A rearrangement was assumed to belong to a leukemic clone either if it had been seen in two amplifications that had originated from two separate aliquots of genomic DNA or if the sequence indicated a lineage relationship to that of a leukemic clone already identified. All rearrangements detected in a sample were amplified from genomic DNA in the one experiment, and the relative abundance of each was calculated using the observed Ct value and the figure of 1.96, as determined experimentally in our laboratory for the amplification factor per PCR cycle. Since estimates of abundance are not precise, we used the following broad criteria to assign a marker to the major, ie, dominant clone or to a minor clone. If there was only one rearrangement that had an abundance of >10%, and its abundance was at least 10-fold greater than that of the next most abundant rearrangement, then this rearrangement was regarded as marking a major clone in which the cells exhibited a monoallelic rearrangement. If there were two or more rearrangements, each with an abundance of >10%, then the two most abundant were regarded as marking the major clone in which the cells exhibited a biallelic rearrangement. All other rearrangements, irrespective of their abundance, were regarded as marking minor clones. In the first study, detection of complete IGH rearrangements by repertoire analysis was compared with that by use of the six V family-specific framework 1 primers and the consensus J primer previously published7van 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: 2257-2317Crossref PubMed Scopus (2518) Google Scholar by the BIOMED-2 consortium. Each amplification reaction used 100 ng of genomic DNA. The end point was the ability to detect complete IGH rearrangements. In the second study, the IGH repertoire was assessed in 50 patients and compared with MRD markers previously identified by the Sydney laboratory. These MRD tests were performed according to the methods and guidelines developed by the BFM MRD task force and the European Study Group on Minimal Residual Disease in Acute Lymphoblastic Leukemia (ESG-MRD-ALL).11van der Velden VH Cazzaniga G Schrauder A Hancock J Bader P Panzer-Grumayer ER Flohr T Sutton R Cave H Madsen HO Cayuela JM Trka J Eckert C Foroni L Zur Stadt U Beldjord K Raff T van der Schoot CE van Dongen JJ European Study Group on MRDdiALL Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data.Leukemia. 2007; 21: 604-611PubMed Google Scholar,12van der Velden VH Panzer-Grumayer ER Cazzaniga G Flohr T Sutton R Schrauder A Basso G Schrappe M Wijkhuijs JM Konrad M Bartram CR Masera G Biondi A van Dongen JJ Optimization of PCR-based minimal residual disease diagnostics for childhood acute lymphoblastic leukemia in a multi-center setting.Leukemia. 2007; 21: 706-713PubMed Google Scholar RQ-PCR quantification was performed for rearrangements of immunoglobulin heavy and κ genes (IGH, IGK) and T-cell receptor δ, δ-α, β, and γ genes (TCRD, TCRD-A, TCRB and TCRG). The identification of mature and immature markers was based on 10 PCR reactions using primers previously published for VDJ and DJ IGH rearrangements.13Verhagen OJ Willemse MJ Breunis WB Wijkhuijs AJ Jacobs DC Joosten SA van Wering ER van Dongen JJ van der Schoot CE Application of germline [email protected] probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia.Leukemia. 2000; 14: 1426-1435Crossref PubMed Scopus (178) Google Scholar The family-specific primers for VH1/7, VH2, VH3, VH4, VH5, VH6, DH2, DH3, and DH6 were tested individually with a common J primer, and a multiplex PCR was used for the rarer DH1, DH4, DH5 and DH7 with the same J primer. For identification of other Ig/TCR markers, family-specific primers were used in singleplex reactions for TCRG, TCRD,6Pongers-Willemse MJ Seriu T Stolz F d'Aniello E Gameiro P Pisa P Gonzalez M Bartram CR Panzer-Grumayer ER Biondi A San Miguel JF van Dongen JJ Primers and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets: report of the BIOMED-1 CONCERTED ACTION: investigation of minimal residual disease in acute leukemia.Leukemia. 1999; 13: 110-118Crossref PubMed Scopus (325) Google Scholar and in multiplex PCRs for TCRB and TCRD-A.7van 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: 2257-2317Crossref PubMed Scopus (2518) Google Scholar,14Szczepanski T van der Velden VH Hoogeveen PG de Bie M Jacobs DC van Wering ER van Dongen JJ Vdelta2-Jalpha Rearrangements are frequent in precursor-B-acute lymphoblastic leukemia but rare in normal lymphoid cells.Blood. 2004; 103: 3798-3804Crossref PubMed Scopus (57) Google Scholar The PCR reactions for five patients for all markers except were performed simultaneously in two 96-well plates with appropriate positive and negative controls. Following PCR, the DNA was heteroduplexed and analyzed on polyacrylamide gels to detect clonal rearrangements15Langerak AW Szczepanski T van der Burg M Wolvers-Tettero IL van Dongen JJ Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations.Leukemia. 1997; 11: 2192-2199Crossref PubMed Scopus (188) Google Scholar that were then verified by sequencing. The number of IGH rearrangements detected in each patient using genomic DNA is shown in Table 2. The median number of complete IGH rearrangements detected per patient was two in children and one in adults (P = 0.016 for age difference, Mann-Whitney test, two-tailed). Rearrangement was not detected in one child and one adult. One rearrangement marking the major clone was detected in 11 children and 13 adults, and two such rearrangements were detected in 13 children and four adults. Repertoire analysis thus detected one or more markers for the major clone in 96% of children and 94% of adults.Table 2Number of Rearrangements Detected Using Genomic DNA in Each Patient in the First Study of 25 Children and 18 AdultsType of rearrangementNone detectedVDJDJNumber of rearrangements01234512Childhood ALL121080121Adult ALL111500001 Open table in a new tab Repertoire analysis on preamplified DNA, also performed in 10 children and 10 adults, detected all previously detected rearrangements, but also detected additional 0 to five rearrangements per patient in both children and adults. These additional rearrangements, 23 in children and 17 in adults, were presumed to mark small minor leukemic clones. Sequencing revealed that, for children, 10 of the 23 minor clones detected were unrelated to the major clone, whereas, for adults, only one of the 17 minor clones detected were unrelated. In two children the rearrangement predominating at diagnosis appeared to have been derived from a founder rearrangement, which was also identified, but at a level of <0.1%. The comparison of repertoire analysis and the BIOMED-2 method for detection of complete IGH rearrangements is shown in Table 3. The repertoire approach detected a greater number of both high-abundance rearrangements (P = 0.03, Fisher's exact test, one-tailed) and low abundance rearrangements (P = 0.001, Fishers exact test, one-tailed) than did the BIOMED-2 approach. The BIOMED-2 approach did not detect seven high-abundance rearrangements owing to concomitant amplification of two rearrangements, which resulted in superimposed and unreadable sequences, and it did not detect rearrangements of intermediate or low abundance. The two rearrangements detected only by the BIOMED-2 approach both involved a pseudogene, which the repertoire analysis primers were not designed to detect.Table 3Number of Rearrangements of High (100–10%), Intermediate (10–1%), or Low (<1%) Abundance Detected by the Repertoire and/or BIOMED-2 Approaches in the First StudyRelative abundance of rearrangement100–10%10–1%<1%Detected by both analyses2926BIOMED-2 only101Repertoire analysis only7123Repertoire analysis was performed using genomic DNA in 15 children and using pre-amplified DNA in 10. The BIOMED-2 analysis was performed in all 25 using genomic DNA. Rearrangements were grouped by their abundance, assessed as the percentage of the total number of rearranged IGH molecules that each rearrangement contributed in a patient. Open table in a new tab Repertoire analysis was performed using genomic DNA in 15 children and using pre-amplified DNA in 10. The BIOMED-2 analysis was performed in all 25 using genomic DNA. Rearrangements were grouped by their abundance, assessed as the percentage of the total number of rearranged IGH molecules that each rearrangement contributed in a patient. The repertoire of IGH rearrangements was determined for 50 clinical trial patients for whom the conventional ESG-MRD-ALL method had been previously used to identify sensitive markers for MRD-based patient risk stratification. The conventional method using family-specific primers identified 206 markers in these patients including 86 IGH rearrangements and 120 rearrangements for IGK, TCRG, TCRD, TCRD-A, and TCRB. Repertoire analysis identified 154 IGH rearrangements. The numbers of IGH rearrangements detected by the two methods are shown in more detail in Table 4. The numbers of complete VDJ and incomplete DJ rearrangements have been pooled and rearrangements have been classified according to whether they mark the major or minor clones and on their abundance. IGH repertoire analysis detected significantly more rearrangements marking both major and minor clones (P < 0.001–0.0001, Fisher's exact test, one-tailed). Factors that appeared to contribute to the difference between the two approaches in the number of IGH rearrangements detected include: for the ESG-MRD-ALL approach, not isolating and sequencing faint PCR products in some cases and not individually sequencing biclonal IGH PCR products unless there were no alternate markers, and for repertoire analysis, the preamplification step that provided sufficient DNA to enable repertoire analysis to then detect rearrangements of intermediate and low abundance.Table 4Number of Rearrangements Detected in 50 Children by the Conventional Method Using Family-Specific Primers and by Repertoire Analysis Using Segment-Specific PrimersClones marked by rearrangementMajor cloneMinor clonesRelative abundance of rearrangement100–10%>10%10–1%1−0.1%<0.1%Detected by both analyses618614Conventional only41010Repertoire analysis only235121024Probability<0.0001NS<0.0001<0.001<0.0001The criteria for major and minor clones are described in Materials and Methods. Rearrangements have been grouped by abundance and the probabilities that the differences in detection of rearrangements arose by chance are shown. NS, not significant. Open table in a new tab The criteria for major and minor clones are described in Materials and Methods. Rearrangements have been grouped by abundance and the probabilities that the differences in detection of rearrangements arose by chance are shown. NS, not significant. When markers for the major clone are considered, both laboratories detected one or more such markers in 48 patients of the 50 patients (96%). The Sydney laboratory found two sensitive rearrangements suitable for patient stratification in 39 patients, one in nine patients, and none in two patients. Since IGH rearrangements enable very sensitive monitoring of MRD by nested PCR, down to 10−6,16Morley AA Latham S Brisco MJ Sykes PJ Sutton R Hughes E Wilczek V Budgen B van Zanten K Kuss BJ Venn NC Norris MD Crock C Storey C Revesz T Waters K Sensitive and specific measurement of minimal residual disease in acute lymphoblastic leukemia.J Mol Diagn. 2009; 11: 201-210Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar we regarded an IGH rearrangement detected by repertoire analysis and marking the major clone as being a marker suitable for sensitive quantification of that clone. Using this criterion, the Flinders laboratory found two such markers in 36 patients, one in 12 patients, and none in two patients. If the results from all of the 75 children studied by repertoire analysis at Flinders are pooled, at least one sensitive marker for the major clone was detected in 96% of children, and two such markers were detected in 65%. For the 50 patients studied by both laboratories, there were 86 IGH markers detected by the Sydney laboratory, but 41 were not used for MRD monitoring b" @default.
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• W2125202362 title "Determining the Repertoire of IGH Gene Rearrangements to Develop Molecular Markers for Minimal Residual Disease in B-Lineage Acute Lymphoblastic Leukemia" @default.
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• W2125202362 doi "https://doi.org/10.2353/jmoldx.2009.080047" @default.
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