Design and Feasibility of a Novel, Rapid, and Simple Fluorescence 26-Plex RT-PCR Assay for Simultaneous Detection of 24 Fusion Transcripts in Adult Acute Myeloid Leukemia
2013; Elsevier BV; Volume: 15; Issue: 2 Linguagem: Inglês
10.1016/j.jmoldx.2012.11.004
ISSN1943-7811
AutoresMarie‐Pierre Laforêt, Pascal Turlure, Éric Lippert, Pascale Cornillet‐Lefèbvre, Arnaud Pigneux, Rachel Pradeau, Jean Feuillard, Nathalie Gachard,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoIdentification of chromosomal abnormalities is mandatory for classification of acute myeloid leukemia (AML), and the abnormalities have to be determined quickly, to allow patient enrollment in multicenter protocols and/or for selecting therapeutic strategies. Rapid AML molecular diagnosis is often difficult to achieve, however, because it is based on numerous different RT-PCR protocols. We developed a new RT-PCR method, one that does not require a nested step, to simultaneously detect all AML fusion transcripts from six major recurrent translocations found in adults: t(15;17)(q22;q12), inv(16)(p13.1q22) [t(16;16)(p13.1;q22)], t(8;21)(q22;q22), t(6;9)(p23;q34), t(9;22)(q34;q11), and t(10;11)(p13;q14). Specific primers for RT-PCR detection of the 24 fusion transcripts, along with two transcripts for controls, were designed for this 26-plex RT-PCR. Each PCR product had a different size and was separated by capillary electrophoresis. We also designed a multiplex positive control with 24 chimeric RNAs, corresponding to all chimeric RNAs tested. Compared with classical molecular biology protocols and cytogenetic analyses used as reference standards, results of the 26-plex RT-PCR method were concordant in all 204 (100%) cases of adult AML tested. Results were obtained in less than 24 hours. Because of the multiplex positive control, interpretation of the peaks was very easy, without any ambiguity. The tumor cell detection threshold was 1.5%. Identification of chromosomal abnormalities is mandatory for classification of acute myeloid leukemia (AML), and the abnormalities have to be determined quickly, to allow patient enrollment in multicenter protocols and/or for selecting therapeutic strategies. Rapid AML molecular diagnosis is often difficult to achieve, however, because it is based on numerous different RT-PCR protocols. We developed a new RT-PCR method, one that does not require a nested step, to simultaneously detect all AML fusion transcripts from six major recurrent translocations found in adults: t(15;17)(q22;q12), inv(16)(p13.1q22) [t(16;16)(p13.1;q22)], t(8;21)(q22;q22), t(6;9)(p23;q34), t(9;22)(q34;q11), and t(10;11)(p13;q14). Specific primers for RT-PCR detection of the 24 fusion transcripts, along with two transcripts for controls, were designed for this 26-plex RT-PCR. Each PCR product had a different size and was separated by capillary electrophoresis. We also designed a multiplex positive control with 24 chimeric RNAs, corresponding to all chimeric RNAs tested. Compared with classical molecular biology protocols and cytogenetic analyses used as reference standards, results of the 26-plex RT-PCR method were concordant in all 204 (100%) cases of adult AML tested. Results were obtained in less than 24 hours. Because of the multiplex positive control, interpretation of the peaks was very easy, without any ambiguity. The tumor cell detection threshold was 1.5%. Diagnosis of acute myeloid leukemia (AML) is made by a combination of different methodologies, including cytology, cytochemistry, immunophenotyping, and cytogenetic and molecular biology, as well as clinical features. Cytogenetic studies of AMLs reveal that 59% of pediatric patients and 52% of adult patients have chromosomal abnormalities.1Raimondi S.C. Chang M.N. Ravindranath Y. Behm F.G. Gresik M.V. Steuber C.P. Weinstein H.J. Carroll A.J. Chromosomal abnormalities in 478 children with acute myeloid leukemia: clinical characteristics and treatment outcome in a Cooperative Pediatric Oncology Group study-POG 8821.Blood. 1999; 94: 3707-3716PubMed Google Scholar, 2Byrd J.C. Mrózek K. Dodge R.K. Carroll A.J. Edwards C.G. Arthur D.C. Pettenati M.J. Patil S.R. Rao K.W. Watson M.S. Koduru P.R. Moore J.O. Stone R.M. Mayer R.J. Feldman E.J. Davey F.R. Schiffer C.A. Larson R.A. Bloomfield C.D. Cancer and Leukemia Group B (CALGB 8461)Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461).Blood. 2002; 100: 4325-4336Crossref PubMed Scopus (1393) Google Scholar Detection of recurrent chromosomal translocations occurring in AMLs is mandatory for correct classification.3Arber D.A. Lenning R.D. Le Beau M.M. Falini B. Vardiman J.W. Porwit A. Thiele J. Bloomfield C.D. Acute myeloid leukaemia with recurrent genetic abnormalities.in: Swerdlow S.H. Campo E. Harris N.L. Jaffe E.S. Pileri S.A. Stein H. Thiele J. Vardiman J.W. WHO Classification of tumors of Haematopoietic and Lymphoid Tissues. IARC Press, Lyon2008: 110-123Google Scholar These chromosomal aberrations are also useful for prognosis stratification of patients among different therapeutic options and sometimes for targeting a specific molecular abnormality.4Mrózek K. Bloomfield C.D. Clinical significance of the most common chromosome translocations in adult acute myeloid leukemia.J Natl Cancer Inst Monogr. 2008; : 52-57Crossref PubMed Scopus (41) Google Scholar, 5Bacher U. Schnittger S. Haferlach T. Molecular genetics in acute myeloid leukemia.Curr Opin Oncol. 2010; 22: 646-655Crossref PubMed Scopus (55) Google Scholar Distribution and frequencies of the known recurrent translocations depend on patient age (Münchner Leukämie Labor, http://www.mll.com/cms/englisch/services/for-physicians/table-of-analyses/table-of-analyses-by-diagnosis/aml-molecular-genetics/MLL, last accessed September 19, 2012). Three recurrent translocations are associated with a favorable prognosis in adult AML: t(15;17)(q22;q12), with three possible fusion transcripts between the PML and RARA genes (bcr1, bcr2, and bcr3), occurs in 8% to 10% of adult AML patients; inv(16)(p13.1q22) [t(16;16)(p13.1;q22)], with 11 possible fusion transcripts between CBFB and MYH11 (types A, B, C, D, E, F, G, H, I, J, and K), occurs in 8% to 12% of adult AML patients; and t(8;21)(q22;q22), with one fusion transcript between the RUNX1 (AML1) and RUNX1T1 (ETO) genes, occurs in 8% to 12% of adult AML patients.6van Dongen J.J. Macintyre E.A. Gabert J.A. Delabesse E. Rossi V. Saglio G. Gottardi E. Rambaldi A. Dotti G. Griesinger F. Parreira A. Gameiro P. Diáz M.G. Malec M. Langerak A.W. San Miguel J.F. Biondi A. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia.Leukemia. 1999; 13: 1901-1928Crossref PubMed Scopus (1011) Google Scholar, 7Park T.S. Lee S.T. Song J. Lee K.A. Lee J.H. Kim J. Lee H.J. Han J.H. Kim J.K. Cho S.R. Choi J.R. Detection of a novel CBFB/MYH11 variant fusion transcript (K-type) showing partial insertion of exon 6 of CBFB gene using two commercially available multiplex RT-PCR kits.Cancer Genet Cytogenet. 2009; 189: 87-92Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar In adult AML, translocations with a poor prognosis are more rare; these include t(6;9)(p23;q34), with one fusion transcript between DEK and NUP214 (CAN) genes, which occurs in 1% of adult AML patients, and inv(3)(q21q26.2) or t(3;3)(q21;q26.2) [RPN1/MECOM (EVI1)], which occurs in 1% of adult AML patients.8Oyarzo M.P. Lin P. Glassman A. Bueso-Ramos C.E. Luthra R. Medeiros L.J. Acute myeloid leukemia with t(6;9)(p23;q34) is associated with dysplasia and a high frequency of flt3 gene mutations.Am J Clin Pathol. 2004; 122: 348-358Crossref PubMed Scopus (80) Google Scholar, 9Medeiros B.C. Kohrt H.E. Arber D.A. Bangs C.D. Cherry A.M. Majeti R. Kogel K.E. Azar C.A. Patel S. Alizadeh A.A. Immunophenotypic features of acute myeloid leukemia with inv(3)(q21q26.2)/t(3;3)(q21;q26.2).Leuk Res. 2010; 34: 594-597Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar The translocation t(9;22)(q34;q11), with four main major-breakpoint cluster region fusion transcripts between the BCR and ABL1 genes (e14a2, e13a2, e14a3, and e13a3), is usually found in chronic myelogenous leukemia (CML), but can also occur in patients with secondary AML or CML in blast crisis (2% of adult AML patients).10Polampalli S. Choughule A. Negi N. Shinde S. Baisane C. Amre P. Subramanian P.G. Gujral S. Prabhash K. Parikh P. Analysis and comparison of clinicohematological parameters and molecular and cytogenetic response of two Bcr/Abl fusion transcripts.Genet Mol Res. 2008; 7: 1138-1149Crossref PubMed Scopus (30) Google Scholar Additionally, the recently identified t(10;11)(p13;q14) is a rare but recurring chromosomal translocation seen in acute lymphoblastic leukemia (ALL) as well as in AML (1% of adult AML patients), resulting in a PICALM (CALM)/MLLT10 (AF10) fusion gene with four possible fusion transcripts between the two genes.11Bohlander S.K. Muschinsky V. Schrader K. Siebert R. Schlegelberger B. Harder L. Schemmel V. Fonatsch C. Ludwig W.D. Hiddemann W. Dreyling M.H. Molecular analysis of the CALM/AF10 fusion: identical rearrangements in acute myeloid leukemia, acute lymphoblastic leukemia and malignant lymphoma patients.Leukemia. 2000; 14: 93-99Crossref PubMed Scopus (75) Google Scholar, 12Borel C. Dastugue N. Cances-Lauwers V. Mozziconacci M.J. Prebet T. Vey N. Pigneux A. Lippert E. Visanica S. Legrand F. Rault J.P. Taviaux S. Bastard C. Mugneret F. Collonges Rames M.A. Gachard N. Talmant P. Delabesse E. Récher C. PICALM-MLLT10 acute myeloid leukemia: a French cohort of 18 patients.Leuk Res. 2012; 36: 1365-1369Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar To name these fusion transcripts (PICALM.1944/MLLT10.241 and PICALM.1944/MLLT10.406 for 5′ transcripts, and PICALM.1944/MLLT10.700 and PICALM.1944/MLLT10.796 for 3′ transcripts), current nomenclature takes the nucleotide position of the breakpoint from the initiation ATG codon of each partner gene, thus reflecting the variety of fusion transcript combinations. Altogether, the six recurrent translocations t(15;17)(q22;q12), inv(16)(p13.1q22) [t(16;16)(p13.1;q22)], t(8;21)(q22;q22), t(6;9)(p23;q34), t(9;22)(q34;q11), and t(10;11)(p13;q14) correspond to 24 different fusion transcripts, often with different breakpoints for the same translocation. Precise molecular diagnosis of adult AML can be a challenging task for laboratories, because a detailed molecular characterization very often has to be given in less than 8 days, to allow patient stratification and enrollment in multicenter therapeutic protocols. At present, many molecular laboratories combine conventional cytogenetics with fluorescence in situ hybridization (FISH), and single RT-PCR; however, FISH is expensive and time consuming, and RT-PCR involves numerous different protocols. At the scale of one hospital center, these resources may be required for very few patients per year. The consensus molecular biology method for such diagnosis is presented in a Concerted Action BIOMED-1 report, which describes up to 12 different single RT-PCRs with their corresponding nested PCRs.6van Dongen J.J. Macintyre E.A. Gabert J.A. Delabesse E. Rossi V. Saglio G. Gottardi E. Rambaldi A. Dotti G. Griesinger F. Parreira A. Gameiro P. Diáz M.G. Malec M. Langerak A.W. San Miguel J.F. Biondi A. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia.Leukemia. 1999; 13: 1901-1928Crossref PubMed Scopus (1011) Google Scholar Attempts have been made to simplify or to accelerate procedures for molecular diagnosis of adult AML. Some studies have been based on electronic hybridization and fluorescence detection or biochips.13Corradi B. Fazio G. Palmi C. Rossi V. Biondi A. Cazzaniga G. Efficient detection of leukemia-related fusion transcripts by multiplex PCR applied on a microelectronic platform.Leukemia. 2008; 22: 294-302Crossref PubMed Scopus (9) Google Scholar, 14Giusiano S. Formisano-Tréziny C. Benziane A. Maroc N. Picard C. Hermitte F. Taranger-Charpin C. Gabert J. Development of a biochip-based assay integrated in a global strategy for identification of fusion transcripts in acute myeloid leukemia: a work flow for acute myeloid leukemia diagnosis.Int J Lab Hematol. 2010; 32: 398-409PubMed Google Scholar Several multiplex RT-PCR systems have been described for detection of some of these fusion transcripts, but often using analysis of the amplified fragments by agarose gel electrophoresis stained with ethidium bromide.15Salto-Tellez M. Shelat S.G. Benoit B. Rennert H. Carroll M. Leonard D.G. Nowell P. Bagg A. Multiplex RT-PCR for the detection of leukemia-associated translocations: validation and application to routine molecular diagnostic practice.J Mol Diagn. 2003; 5: 231-236Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 16Pakakasama S. Kajanachumpol S. Kanjanapongkul S. Sirachainan N. Meekaewkunchorn A. Ningsanond V. Hongeng S. Simple multiplex RT-PCR for identifying common fusion transcripts in childhood acute leukemia.Int J Lab Hematol. 2008; 30: 286-291Crossref PubMed Scopus (33) Google Scholar For example, Pallisgaard et al17Pallisgaard N. Hokland P. Riishøj D.C. Pedersen B. Jørgensen P. Multiplex reverse transcription-polymerase chain reaction for simultaneous screening of 29 translocations and chromosomal aberrations in acute leukemia.Blood. 1998; 92: 574-588PubMed Google Scholar proposed a protocol based on eight multiplex RT-PCRs for screening of 29 acute leukemia chromosomal translocations known at the time of the publication; however, these orientation multiplex RT-PCRs had to be followed by different nested PCRs, specific for the suspected fusion transcripts, and had to be confirmed by sequencing. To our knowledge, to date none of the published techniques detect these six translocations together in a simple multiplex RT-PCR and using capillary electrophoresis. Here, we describe a rapid and reproducible method to simultaneously detect 24 fusion transcripts using a 26-plex RT-PCR method. Each PCR product had a different size, and all PCR products could be separated by capillary electrophoresis. We also developed a multiplex positive control that includes all tested fusion transcripts; it is produced by in vitro transcription, controls all steps in the procedure after RNA purification, and provides a marker for the position of each PCR product after capillary electrophoresis. After approval of the Institutional Review Board of the Limousin Region (Direction de la Recherche Clinique et Comité de Protection des Personnes du Limousin) and with informed oral consent (ie, absence of written opposition), 204 patients from university hospitals in France (Limoges, 175 cases; Bordeaux, 19 cases; Reims, 10 cases) were retrospectively included at diagnosis between 2004 and 2012. Residual bone marrow or blood samples were obtained in the normal course of patient staging without any additional sampling and after all techniques needed for diagnosis of their pathology had been performed. This series included 87 patients (42.6%) with one of the six studied translocations. Among these, 41/204 (20.1%) had a diagnosis of acute promyelocytic leukemia (APL). Limoges center is a reference center for the molecular follow-up of APL in France, which accounts for the frequency of APL recruitment. Diagnosis of AML was established according to standard morphological and cytochemical criteria, as well as to immunophenotypic, molecular, and cytogenetic results. Total RNA was extracted either using a QIAmp RNA blood mini kit (Qiagen, France) or after lysis and storage in TRIzol reagent solution according to the manufacturer's recommendations (Life Technologies–Invitrogen, Carlsbad, CA; Saint Aubin, France). Detection of fusion transcripts was performed at diagnosis for all patients, using the simplex RT-PCR series described in the BIOMED-1 protocol and according to methods described previously.6van Dongen J.J. Macintyre E.A. Gabert J.A. Delabesse E. Rossi V. Saglio G. Gottardi E. Rambaldi A. Dotti G. Griesinger F. Parreira A. Gameiro P. Diáz M.G. Malec M. Langerak A.W. San Miguel J.F. Biondi A. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia.Leukemia. 1999; 13: 1901-1928Crossref PubMed Scopus (1011) Google Scholar, 7Park T.S. Lee S.T. Song J. Lee K.A. Lee J.H. Kim J. Lee H.J. Han J.H. Kim J.K. Cho S.R. Choi J.R. Detection of a novel CBFB/MYH11 variant fusion transcript (K-type) showing partial insertion of exon 6 of CBFB gene using two commercially available multiplex RT-PCR kits.Cancer Genet Cytogenet. 2009; 189: 87-92Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 18Nakamura F. Maki K. Arai Y. Nakamura Y. Mitani K. Monocytic leukemia with CALM/AF10 rearrangement showing mediastinal emphysema.Am J Hematol. 2003; 72: 138-142Crossref PubMed Scopus (7) Google Scholar RNA was also extracted from five different human leukemia/lymphoma cell lines with specific translocations: NB4 for the PML/RARA bcr1 transcript, ME-1 for the CBFB/MYH11 type A transcript, Kasumi-1 for the RUNX1/RUNX1T1 transcript, K562 for the BCR/ABL1 e14a2 transcript, and U937 for the PICALM.1944/MLLT10.241 5′ transcript. The plasma cell line L-363 was used for the threshold detection determination. All cell lines were purchased from DSMZ (Germany). To establish a multiplex reverse transcription and cDNA amplification method for 26 transcripts (26-plex RT-PCR), a set of primers specific for each fusion transcript and two gene controls was designed to obtain each PCR product at a specific size and thus allow us to assign each size to a specific fusion transcript on the electrophoretic profile. The cDNA specific primer sequences used in these assays are listed in Table 1. Primers were designed using GenomeLab GeXP eXpress Designer software version 1.0 (Beckman Coulter, Brea, CA; Krefeld, Germany) and were evaluated by BLAST searches to ensure a specific amplification of the designed PCR fragments. Quality of primers was checked using Vector NTI Advance software version 11.0 (Life Technologies–Invitrogen). Tag1 sequence (5′-GTACGACTCACTATAGGGA-3′) was added to the 5′ end of all reverse cDNA specific primers, and Tag2 sequence (5′-AGGTGACACTATAGAATA-3′) was added to the 5′ end of all forward cDNA specific primers. With these tagged specific primers, the predicted sizes of fragments ranged from 120 to 704 bp (Table 1). Assignment of PCR size products to each fusion transcript took into account between-patient variability in breakpoints. For example, the PML/RARA bcr2 fusion transcript corresponds to a variable breakpoint in the 3′ part of exon 6 of PML, with, as a consequence, unpredictable sizes of amplification products. We therefore designed the corresponding set of primers so that both bcr1 and bcr2 PML/RARA fusion transcripts could be amplified and also so that, regardless of the position of the breakpoint, amplification of the PML/RARA bcr2 fusion transcript would lead to the smallest PCR product and would always be smaller than the bcr1 transcript (Table 1).Table 1Translocations, Transcripts, Primers, and Expected versus Observed Product Sizes for 26-Plex RT-PCR for Simultaneous Detection of AML Fusion TranscriptsTranslocationFusion transcriptForward primer 5′ Tag2-seq 3′Reverse primer 5′ Tag1-seq 3′RT-PCR product (bp)ExpectedObservedt(15;17)(q22;q12)PML/RARA bcr15′-ACCTGGATGGACCGCCTA-3′5′-AGGGCTGGGCACTATCTCTT-3′180179PML/RARA bcr2SameSame156157PML/RARA bcr35′-GTATCCTGCTGGAGGCTGTGGAC-3′Same384377inv(16)(p13.1q22)[t(16;16)(p.13.1;q22)]CBFB /MYH11 type A5′-ATCGGGCTGTCTGGAGTTTGATG-3′5′-CTTGAGCGCCTGCATGTT-3′311311CBFB /MYH11 type BSameSame524524CBFB /MYH11 type CSameSame704707CBFB /MYH11 type FSameSame215215CBFB /MYH11 type DSame5′-CTGGTCTTGCAGGCTGTTC-3′365364CBFB /MYH11 type ESameSame572569CBFB /MYH11 type GSameSame269268CBFB /MYH11 type HSameSame372369CBFB /MYH11 type JSameSame260259CBFB /MYH11 type KSameSame461458CBFB /MYH11 type ISame5′-GTCCTGGAGTGCGTTCCTT-3′471469t(8;21)(q22;q22)RUNX1/RUNX1T15′-TCGAAGTGGAAGAGGGAAAA-3′5′-GCCATTCAAGGCTGTAGGAG-3′335335t(9;22)(q34;q11)BCR/ABL1 type e14a25′-CAGATGCTGACCAACTCGTG-3′5′-AGCAGATACTCAGCGGCATT-3′525530BCR/ABL1 type e13a2SameSame450453BCR/ABL1 type e14a3SameSame351353BCR/ABL1 type e13a3SameSame276277t(10;11)(p13;q14)PICALM.1944/MLLT10.2415′-TGAGACCTCCAAACCCCTTT-3′5′-TCGGCACCATTACCTTCTTC-3′395398PICALM.1944/MLLT10.406SameSame230231PICALM.1944/MLLT10.700Same5′-AAATCCTGGGGAGACTGCAC-3′500501PICALM.1944/MLLT10.796SameSame404405t(6;9)(p23;q34)DEK/NUP2145′-AAGTTGAAGAAACCCCCTACAGA-3′5′-ATCATTCACATCTTGGACAGCA-3′196196ABL1 control5′-GCCCCCGTTCTATATCATCA-3′5′-AGCAGATACTCAGCGGCATT-3′296298B2M control5′-GGCATTCCTGAAGCTGACA-3′5′-CTAAGTTGCCAGCCCTCCTA-3′668681Tag2 and Tag1 sequences were added to the 5′ end of Forward and Reverse specific primers respectively. A spacer sequence, indicated by bolded nucleotides, was added between Tag2 and forward specific sequences for the PML/RARA bcr3 fusion transcript, as well as for all CBFB/MYH11 fusion transcripts. Gene accession numbers: PML: NM_033244.3, RARA: NM_000964.3, CBFB: NM_022845.2, MYH11: NM_002474.2,RUNX1: NM_001754.4, RUNX1T1: NM_175635.2, BCR: NM_004327.3, ABL1: NM_007313.2, PICALM: NM_007166.2, MLLT10: NM_004641.3, DEK: NM_003472.3, NUP214: NM_005085.2, B2M: NM_004048.2. Open table in a new tab Tag2 and Tag1 sequences were added to the 5′ end of Forward and Reverse specific primers respectively. A spacer sequence, indicated by bolded nucleotides, was added between Tag2 and forward specific sequences for the PML/RARA bcr3 fusion transcript, as well as for all CBFB/MYH11 fusion transcripts. Gene accession numbers: PML: NM_033244.3, RARA: NM_000964.3, CBFB: NM_022845.2, MYH11: NM_002474.2,RUNX1: NM_001754.4, RUNX1T1: NM_175635.2, BCR: NM_004327.3, ABL1: NM_007313.2, PICALM: NM_007166.2, MLLT10: NM_004641.3, DEK: NM_003472.3, NUP214: NM_005085.2, B2M: NM_004048.2. An internal control for the RT-PCR reaction is included in the Beckman Coulter GenomeLab GeXP start kit, consisting of a kanamycin RNA (Kanr) that contains an RNase inhibitor, along with its specific tagged primers. The expected size of the PCR product of the Kanr control is 325 bp. Four primers for two positive controls were added to the assay, to assess the quality of RNA samples, targeting ABL1 and B2M as reference genes. The expected amplification product size was 296 bp for ABL1 (to validate medium size RT-PCR amplicons) and 668 bp for B2M (to validate large amplification products). A multiplex positive control was designed to check, in another reaction tube, that the 26-plex RT-PCR gave rise to the intended PCR products with correct sizes for each of the 24 fusion transcripts sought. To obtain this pool of RNAs, either synthetic DNAs (11 fusion transcript sequences, purchased from Eurofins Medigenomix, Ebersberg, Germany) or cDNAs obtained from reverse transcription of fusion transcripts from patients or cell lines with specific chromosome translocation (13 fusion transcripts), as well as cDNA for the ABL1 and B2M reference genes, were cloned into the TOPO TA pCR2.1 plasmid (Life Technologies–Invitrogen), which allows in vitro transcription. After RNA purification, the multiplex positive control was thus a mix of the 24 chimeric RNA species and two RNA reference genes, each obtained after in vitro transcription with a Riboprobe System T7 kit (Promega, Madison, WI; Lyon, France) and diluted to an adequate concentration to obtain a fluorescence signal in the same range of scale after capillary electrophoresis. This multiplex positive control was used in a supplementary reaction tube, instead of patient RNA, to validate all reagents in the RT-PCR multiplex and to give all transcript sizes. The 26-plex reverse transcription reactions were simultaneously performed using a Beckman Coulter GenomeLab GeXP start kit in a 20-μL reaction volume with 100 ng total RNA from patients or cell lines, or with 0.5 ng of multiplex positive control (concentration of each RNA species ranged from 0.5 to 65 pg/μL), and 1 ng Kanr RNA control. The final concentration of each specific reverse primer with the additional Tag1 sequence (Tag1 specific primer) was adjusted to 50 nmol/L, except for Tag1 primers specific for RUNX1/RUNX1T1 and ABL1 control transcripts, which were adjusted to 12.5 nmol/L final, and Tag1 primers specific for B2M control adjusted to 5 nmol/L final. Enzymatic reaction was performed at 48°C for 1 minute, 42°C for 60 minutes, and was followed by a denaturation step at 95°C for 5 minutes. All 26-plex PCR reactions were performed in a 20 μL reaction volume using 9.3 μL of each Reverse Transcription reaction and Tag2 forward specific primers at a final concentration of 20 nmol/L each, 3.5 units Thermo-Start Taq polymerase (ABgene; Thermo Scientific, Waltham, MA; Illkirch, France), 5 mmol/L final MgCl2 (ABgene; Thermo Scientific), and 1× final of PCR buffer provided in the Beckman Coulter GenomeLab GeXP start kit (which includes both 5′ and 3′ universal primers complementary to Tag1 and Tag2 sequences). DNA amplification cycles were an initial 95°C for 10 minutes for activation of the Taq and denaturation of DNA samples, then three cycles at 94°C for 30 seconds, 60°C for 30 seconds, and 70°C for 2 minutes, and finally 32 cycles at 94°C for 30 seconds, 55°C for 30 seconds, and 70°C for 2 minutes. Concentration of specific primers coupled to Tag1 and Tag2 were established to favor amplification from cDNAs for the first three cycles. Then, because of the high concentration of the 5′ and 3′ universal primers and the annealing temperature chosen, DNA synthesis was initiated mainly from both 5′ and 3′ universal sequences for the next 32 cycles. The 5′ universal WellRED D4-PA dye labeled primer was included in the GenomeLab GeXP start kit and allowed detection of amplified fragments by capillary electrophoresis. The principles of the 26-RT-PCR protocol are presented schematically in Supplemental Figures S1 and S2. Two micoliters of each PCR product was added to 0.25 μL of Beckman Coulter GenomeLab DNA size standard 600 and 37.75 μL of a sample loading solution provided in the GenomeLab GeXP start kit. Samples were subjected to capillary electrophoresis using a CEQ 8800 genetic analysis system (Beckman Coulter) and were processed as follows: capillary temperature at 50°C, denaturation at 90°C for 120 seconds, injection at 2 kV for 30 seconds, and separation at 4.8 kV for 95 minutes. Analysis of fragment size was performed with a Beckman Coulter GenomeLab GeXP genetic analysis system. Our aim was to design a unique multiplex RT-PCR (in our case, a 26-plex RT-PCR) to detect the 24 fusion transcripts of six important recurrent chromosomal translocations useful for prognosis in adult AML: t(15;17)(q22;q12), inv(16)(p13.1q22) [t(16;16)(p13.1;q22)], t(8;21)(q22;q22), t(6;9)(p23;q34), t(9;22)(q34;q11), and t(10;11)(p13;q14). The technology was based on the design of reverse and forward primers specific for each of the mRNA species to be detected,19Drew J.E. Mayer C.D. Farquharson A.J. Young P. Barrera L.N. Custom design of a GeXP multiplexed assay used to assess expression profiles of inflammatory gene targets in normal colon, polyp, and tumor tissue.J Mol Diagn. 2011; 13: 233-242Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar with added universal Tag1 and Tag2 sequences, as described under Materials and Methods. With this method, the first cycles of amplification were from specific sequence templates. The amplification started from the Tag1 and Tag2 added sequences, because of the universal primers, the forward one being tagged with WellRED D4-PA dye (Supplemental Figures S1 and S2). Identification of a specific species among the 24 possible chimeric RNAs was based on size differences of amplification products. Primers were designed to give a difference of ≥5 bp between each peak. However, the observed size of some PCR products differed slightly from the theoretical expected size. This led to peak overlaps between transcript K of CBFB/MYH11 and transcript e13a2 of BCR/ABL1 (Figure 1A). To better separate the genuine observed peaks from each mRNA species, an atc or a gtatc spacer sequence was inserted between the Tag2 sequence and the forward specific primer for CBFB/MYH11 fusion transcripts and the PML/RARA bcr transcript, respectively (Table 1). With the spacer sequences, separation between the different peaks reached ≥5 bp (Figure 1B). Next, we designed a multiplex positive control containing each of the targeted RNA species that can be used in a single tube to provide a size marker for each fusion transcript. Each transcript was correctly separated and could be easily detected; that is, each transcript could be specifically assigned to a single corresponding peak size (Figure 2). Reproducibility was tested on 70 RT-PCR reactions of the multiplex positive control. For each RT-PCR, the 24 peaks (as well as peaks for the ABL1, B2M, and Kanr controls) were identified at the same size (data not shown). To estimate the detection efficiency of the technique, dilution experiments were performed with the Kasumi-1 and K562 cell lines, respectively bearing the t(8;21)(q22;q22) translocation (RUNX1/RUNX1T1 fusion transcript, 335-bp amplification product) and the t(9;22)(q34;q11) (BCR/ABL1 fusion transcript, 530-bp amplification product), respectively, (Figure 3A). The detection threshold was defined as background + 2 SD. Blast cell detection with a specific fusion transcript was effective down to a threshold of 1.5% (where 100% corresponds to 100 ng of starting RNA) (Figure 3B). Characterization of the cytogenetic and molecular status of patients was performe
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