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A novel nested-PCR strategy for the detection of rearranged immunoglobulin heavy-chain genes in B cell tumors

A novel nested-PCR strategy for the detection of rearranged immunoglobulin heavy-chain genes in B cell tumors
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  Leukemia (1997) 11 , 1793–1798 󰂩  1997 Stockton Press All rights reserved 0887-6924/97 $12.00 BTS TECHNICAL REPORT BTS  Leukemia  A novel nested-PCR strategy for the detection of rearranged immunoglobulin heavy-chain genes in B cell tumors C Voena 1 , M Ladetto 1 , M Astolfi 1 , D Provan 2 , JG Gribben 2 , M Boccadoro 1 , A Pileri 1 and P Corradini 1 1 Dipartimento di Medicina ed Oncologia Sperimentale, Divisone Universitaria di Ematologia, Azienda Ospedaliera San Giovanni Battista, Torino,Italy; and   2 Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA Several methods have been developed for the detection of  In B cell malignancies, several methods have been minimal residual disease (MRD) in B cell tumors. Chromosomal developed for the detection of MRD. 9 Tumor-specific break- translocations or the rearrangement of the immunoglobulin points of chromosomal translocations and clonal rearrange- heavy chain (IgH) and T cell receptor genes are generally ments of immunoglobulin heavy chain (IgH) genes are used employed. We report a novel PCR method to detect MRD using as molecular markers. 4,5,10,11 The chromosomal translocations IgH genes. IgH rearranged variable region (VDJ) were amplifiedfrom tumor specimens using consensus primers for variable  most frequency employed are the t(14;18) present in about and joining region genes. Complementarity-determining 50% of B cell non-Hodgkin’s lymphomas (NHL), the t(9;22) regions (CDR) were identified and used to generate tumor-spe- present in 5% of childhood and 30% of adult acute lympho- cific primers. Two-round amplifications using primers derived blastic leukemias (ALL), the t(1;19) in 25% pre-B ALL, the from CDRs and joining or constant regions were performed for t(4;11) in 2–5% ALL and the recently described t(12;21) in MRD detection. IgH nested-PCR approach was tested on apanel of 75 B cell tumors including acute lymphoblastic and  25% childhood precursor B-ALL. 5,12–16 Moreover, chromo- chronic lymphocytic leukemias, non-Hodgkin’s lymphomas somal translocations can be employed only in a subset of  and multiple myelomas. A VDJ sequence was obtained in 62 patients with B cell malignancies whereas the rearrangment outof 75 cases (83%). Sensitivityusing DNAor cDNA templates of IgH genes may provide a tumor marker in about 90% of  was 10 − 5 and  − 6 , respectively. Thismethod is specific and sensi- patients since it is a lineage-specific marker. tive and provides a simple, non-radioactive approach for theevaluation of MRD in B cell tumors.  During B cell ontogeny, the genes that encode for IgH Keywords:  B cell malignancies; IgH nested-PCR; minimal undergo rearrangement of variable (V), diversity (D) and join- residual disease ing (J) segments, to generate antigen-specific receptors. Therearranged variable region (VDJ) is composed of four relativelyconserved regions, called framework (FR), interrupted by three Introduction complementarity-determining regions (CDR). CDRs codify forthe antigen-binding site and are unique for each B cellB cell malignancies are a heterogeneous group of disordersclone. 17 In B cell malignancies, CDRs are utilized to designcharacterized by the proliferation of clonal cells at variouspatient-specific primers and probes for the amplification andstages of differentiation. In recent years, intensified chemo-hybridization procedures for MRD detection. 11,18 Althoughradiotherapy programs followed by autologous or allogeneicsuch procedures are sensitive and specific, the hybridizationbone marrow transplantation have been successfullystep is expensive, time-consuming and requires the use of aemployed for the treatment of these tumors. Although com-radioactive labeled probe. Nested-PCR has been recently usedplete remissions (CR) are often achieved, disease relapsefor MRD detection in order to bypass the hybridization step.remains the leading cause of death in these patients. 1–3 Nested-PCR is a simple nonradioactive procedure which isThe analysis of minimal residual disease (MRD) hascurrently used when tumor-specific translocations are present,assumed a growing role in the evaluation of tumor cell con-providing the same sensitivity and specificity of amplificationtamination of autografts and during molecular follow-up of hybridization procedures. 19 patients otherwise in CR. 4–6 It has recently been shown thatIn the present study, we show a nested-PCR approach forpatients with chronic myelogenous leukemia, acutethe detection of MRD using IgH genes. Double amplificationpromyelocytic leukemia, acute lymphoblastic leukemia or fol-using specific primers derived from tumor CDRs (CDRII andlicular non-Hodgkin’s lymphoma may remain PCR-positiveCDRIII) has been employed. Our approach has been testedafter chemotherapy or transplantation procedures. The persist-on a panel of 75 B cell tumors including seven ALL, 22 NHL,ence of such a positivity has been correlated with a highernine chronic lymphocytic leukemia (CLL) and 37 multiplefrequency of relapse. 5–8 myeloma (MM). Correspondence: C Voena, Cattedra di Ematologia, Via Genova 3,10126 Torino, ItalyReceived 17 February 1997; accepted 16 June 1997  BTS Technical Report C Voena  et al  1794 Table 1  Amplification of the tumor VDJ primers derived from the leader region VH.LB  a VH.LS  b VH1 5 ′ -CCATGGACTGGACCTGGAGG-3 ′  VH1 5 ′ -CTCACCATGGACTGGACCTGGAG-3 ′ VH2 5 ′ -ATGGACATACTTTGTTCCAG-3 ′  VH2 5 ′ -ATGGACATACTTTGTTCCACGCTC-3 ′ VH3 5 ′ -CCATGGAGTTTGGGCTGAGC-3 ′  VH3 5 ′ -CCATGGAGTTTGGGCTGAGCTGG-3 ′ VH4 5 ′ -ATGAAACACCTGTGGTTCTT-3 ′  VH4 5 ′ -ACATGAAACA(C/T)CTGTGGTTCTTCC-3 ′ VH5 5 ′ -ATGGGGTCAACCGCCATCCT-3 ′  VH5 5 ′ -ATGGGGTCAACCGCCATCCTCCG-3 ′ VH6 5 ′ -ATGTCTGTCTCCTTCCTCAT-3 ′  VH6 5 ′ -ATGTCTGTCTCCTTCCTCATCTTC-3 ′ a See Ref. 22. b See Ref. 23. Materials and methods  primer derived from the JH 3 ′  end (JH3 5 ′ -ACCTGAGGA-GACGGTGACCAGGGT-3 ′ ). The reaction was carried out for33 cycles (denaturation 94 ° C for 30 s, annealing 65 ° C for 30 s Patients and nucleic acid extraction  and extension 72 ° C for 30 s) with a final extension of 7 min.The prevention of PCR contamination was performed as pre-Seventy-five patients with B cell tumors were selected for thisstudy. PB, BM and lymph node samples were obtained during viously described. 25 PCR products were analyzed by electro-phoresis on 2% agarose gel. Direct sequencing of amplifiedstandard diagnostic procedures. They had different degrees of tumor infiltration, ranging from 2 to 90%. BM and/or PB cells DNAs was performed using the Promega fmol sequencing sys-tem as previously described. 26 Reactions were carried out inwere separated on a Ficoll–Hypaque density gradient. DNAwas obtained by cell lysis, phenol extraction and ethanol pre- a thermocycler at 68 ° C annealing temperature for 15 cycles.When the sequence quality did not allow a complete readingcipitation. 20 RNA was isolated using RNAzol B method(Biotex Laboratories, Houston, TX, USA). Total cDNA syn- of CDR regions, DNA was reamplified with primers contain-ing  Eco  RI and  Hin  dIII restriction sites, and cloned in athesis was carried out as follows: total RNA (5   g) wasreverse-transcribed with 20 pmol of reverse transcription Bluescript SK vector (Stratagene, San Diego, CA, USA). 20 Restriction enzyme analysis was carried out on plasmid DNAsprimer (C  1: 5 ′ -AAGGAAGTCCTGTGCGAGG-3 ′ , C   1: 5 ′ -GGAAGTAGTCCTTGACCAG-3 ′ , C  1: 5 ′ -AGAAGCCCT- prepared by the alkaline lysis method, and miniprep plasmidDNAs were then sequenced. The analysis of sequencing wasGGACCAGGCA-3 ′ ). A 50-  l reaction was performed in10 mmol/l dithiothreitol, 1 mmol/l deoxynucleoside triphos- performed using the PC-GENE software (IntelliGenetics,Mountain View, CA, USA).phates (dNTPs; Pharmacia LKB Biotechnology, Uppsala,Sweden), 1  reverse transcriptase buffer (50 mmol/l Tris-HCl,6 mmol/l MgCl 2 , 40 mmol/l KCl) final concentration, adding20 U of ribonuclease inhibitor (RNasin; Promega, Madison,  Detection of residual tumor cells  WI, USA) and 200 U Moloney murine leukemia virus reversetranscriptase (Superscript; GIBCO BRL, Gaithersburg, MD, Nested-PCR for IgH rearrangement was carried out as follows:for DNA samples, 1  g of genomic DNA was amplified usingUSA). The reaction mixture was incubated for 1 h at 37 ° C.After cDNA synthesis, reverse transcriptase was heat-inacti- a consensus primer derived from the tumor VH family and anantisense derived from the 3 ′  end of the JH region (JH3). Thevated for 5 min at 95 ° C.first reaction was carried out for 28 cycles (denaturation 94 ° Cfor 30 s, annealing 64 ° Cfor 30 s, extension 72 ° Cfor 30 s) witha final extension of 7 min. One   l of amplified DNA was then Amplification and sequencing of the tumor VDJ  reamplified using CDRII sense and CDRIII antisense primersusing 2.5 U of AmpliTaq Gold (PE Applied Biosystems, RocheVDJs were amplified starting from genomic DNA or totalcDNA depending on sample availability. Amplifications were Molecular Systems, Branchburg, NJ, USA). The second reac-tion was carried out for 35 cycles (denaturation 94 ° C for 30 s,performed as previously described. 18 Briefly, 1   g of genomicDNA or 1   l of total cDNA, was amplified using two sets of annealing depending on specific primers melting temperature( T  m ) for 30 s, extension 72 ° C for 30 s) with a final extensionconsensus sense primers derived from the IgH leader regionand FR1 respectively 21–24 (Tables 1 and 2), and an antisense of 7 min. For cDNA samples, 1   l of cDNA (prepared from Table 2  Amplification of the tumor VDJ primers derived from the first framework region VH.FD  a VH.FS  b VH1 5 ′ CCTCAGTGAAGGTCTCCTGCAAGG-3 ′  VH1 5 ′ -CAGGTGCAGCTGGTGCA(C/G)(A/T)CTG-3 ′ VH2 5 ′ -TCCTGCGCTGGTGAAAGCCACACA-3 ′  VH2 5 ′ -CAG(A/G)TCACCTTGAAGGAGTCTG-3 ′ VH3 5 ′ -GGTCCCTGAGACTCTCCTGTGCA-3 ′  VH3 5 ′ -CAGGTGCAGCTGGTG(C/G)AGTC(C/T)G-3 ′ VH4a 5 ′ -TCGGAGACCCTGTCCCTCACCTGCA-3 ′  VH4a 5 ′ -GAG(C/G)TGCAGCTGCAGGAGTC(C/G)G-3 ′ VH4b 5 ′ -CGCTGTCTCTGGTTACTCCATCAG-3 ′  VH4b 5 ′ -CAGGTGCAGCTACA(A/G)CAGTGGG-3 ′ VH5 5 ′ -GAAAAAGCCCGGGGAGTCTCTGAA-3 ′  VH5 5 ′ -GAGGTGCAGCTG(G/T)TGCAGTCTG-3 ′ VH6 5 ′ -CCTGTGCCATCTCCGGGGACAGTG-3 ′  VH6 5 ′ -CAGGTACAGCTGCAGCAGTCAG-3 ′ a See Ref. 21. b See Ref. 20.  BTS Technical Report C Voena  et al  1795 5  g of total RNA as described above) was amplified using formed as described in the Materials and methods section,showed that the most common VH family used was VH3CDRII primer with an antisense primer derived from the firstexon of the IgH tumor constant region (CH1, the same primer (61%) for all B cell malignancies studied, in particular 57%of ALL, 50% of NHL, 49% of MM and 55% of CLL. The mostused in RT reaction). Amplification was carried out for 28cycles (denaturation 94 ° C for 30 s, annealing depending on represented JH was JH4, especially in MM (49%) and NHL(36%), followed by JH6 (29% in ALL, 19% in MM, 33% inspecific primer  T  m , extension 72 ° Cfor 30 s). Second PCR reac-tion was performed using a primer derived from CDRIII and CLL and 9% in NHL). Tumor-specific primers for nested-PCRto detect MRD were derived from CDRs sequences. For eachan internal antisense primer derived from the IgH constantregion (CH2) (C  2: 5 ′ -CAGGAGACGAGGGGGAAA-3 ′ , C   2: patient CDRII sequence was used to synthesize a senseprimer, while the CDRIII sequence was used to synthesize5 ′ -CAGAGGTGCTCCTGGAGCA-3 ′ , C  2: 5 ′ -ACCACG-TTCCCATCTGGCTG-3 ′ ). The first and the second reactions either a sense or an antisense primer as described in theMaterials and methods section.were performed using AmpliTaq Gold (PE Applied Biosystems)Specific primers for the second reaction were selected, whenpossible, with a slightly higher melting temperature to minim-ize inappropriate annealing of primers used during the first  Double strategy to detect MRD with IgH nested-PCR  amplification. PCR products were visualized on a 3% Meta-phor agarose gel (FMC Bioproducts, Rockland, ME, USA) Nested-PCR using CDRII and CDRIII primers was successfullyperformed in all patients in which VDJ sequence was avail-stained with ethidium bromide (0.5  g/ml). Two polyclonalDNAs were always used as negative controls. able. For each patient, the specificity of the method wasevaluated by amplifying two polyclonal DNAs under the sameSensitivity experiments were performed diluting both celllines and tumor cells from patients (with at least 90% of circul- conditions of tumor DNA and no amplifications wereobserved. In order to determine whether the amplification forating tumor cells) with normal marrow cells (up to one tumorcell in 10 6 normal marrow cells). Cell mixtures were pelleted MRD detection is more efficient when performed on RNAwith an RT-nested PCR based approach or on genomic DNAand resuspended in 10   l of H 2 O and then lysed by heatingat 95 ° C for 5 min with 10 U of ribonuclease inhibitor (RNasin; with a simple nested-PCR, two experimental strategies wereadopted and compared. Both methods were used dependingPromega). The whole lysate from each serial dilution wasamplified with specific nested primers for DNA strategy, on sample availability. On DNA samples the strategy, calledDNA strategy, was based on a first amplification with a senseotherwise reverse transcribed into cDNA in a total volume of 20  l. The whole cDNA was then amplified following the pre- primer derived from patient clonal VH family and an antisenseprimer derived from the 3 ′  end of the JH region. Samples wereviously described conditions in a 100   l final volume.then reamplified with patient-specific CDR primers: CDRIIsense and CDRIII antisense primers (Figure 2). On RNAsamples the approach, called cDNA strategy, involved a Oligonucleotide synthesis  reverse transcription reaction to obtain tumor-specific IgHcDNA followed by a two-round amplification. Both amplifi-Oligonucleotides were chemically synthesized with a 391PCR-MATE EP, DNA synthesizer (Applied Biosystems, Foster cations were performed with 5 ′  sense primers derived frompatient-tumor CDRs (CDRII and CDRIII) and 3 ′  antisense pri-City, CA, USA) on a 0.2   mol scale, according to the users’manual. mers derived from the first exon of the tumor IgH constantregion (Figure 3). All the patients examined gave a band of the expected size. No false positive results were observed inany strategy. Overall, we could perform both strategies on 40 Results patients (31 MM, seven NHL, two CLL). DNA strategy wasused for all the ALL patients as most of them do not express Identification of VDJ sequence  the    chain in the cytoplasm. DNA strategy was also usedwhen only DNA samples were available (two MM, 10 NHL,VDJ regions were amplified using two sets of 5 ′  consensusprimers derived from the leader or FR1 region of IgH genes four CLL). In order to compare the sensitivity of both strategies,two amplifications were performed on serial dilutions onand a 3 ′  primer from the JH region. First, specimens wereamplified with 5 ′  consensus primer from FR1 and tumor VDJ either DNA or RNA of a Burkitt lymphoma cell line (BJAB).The sensitivity was higher with the cDNA strategy as itwas obtained by direct sequencing in 31 patients; thenremaining amplified VDJs were cloned in a plasmid vector. allowed the detection of one tumor cell in 10 6 normal marrowcells, while the DNA strategy reached the sensitivity of oneFor each patient 10 to 15 clones were sequenced and a pre-dominant clone was found in 15 patients. Alternatively, those tumor cell in 10 5 normal marrow cells. Sensitivity experimentswere also performed on 10 patients, diluting tumor cells withpatients whose VDJ sequence was impossible to obtain withFR1 amplification, were amplified with 5 ′  consensus primer normal marrow cells. The results were similar to thoseobtained with the cell line: a 10 times lower sensitivity wasfrom the leader region and then sequenced directly or cloned.Tumor VDJ was obtained by direct sequence only in five usually obtained with DNA strategy. We also compared thesedata with the results previously obtained in 55 patients usingpatients, while cloning and sequencing was successful in 11patients. Overall, complete sequence of the tumor VDJ was the hybridization procedure: the tumor clone was amplifiedwith CDRII and JH or CH primers and then hybridized to aobtained in 62 patients out of 75 (83%) (77% of NHL, 86%of ALL, 66% of CLL and 89% of MM patients) (Figure 1). Prob- CDRIII probe. IgH nested-PCR results were identical showingthe same sensitivity and specificity of the hybridizationably, either the presence of somatic mutations or the use of unknown variable regions were the cause of the lack of ampli- procedure.An alternative approach suitable for DNA and RNA tem-fication in 13 patients. There was no correlation between asuccessful amplification of VDJ region and the number of plates was also performed, based on a nested-PCR with senseprimers derived from the tumor CDRs (CDRII and CDRIII) andtumor cells in the diagnostic sample. Sequence analysis, per-  BTS Technical Report C Voena  et al  1796 Figure 1  Strategy used to amplify and sequence CDRII and CDRIII regions from 75 patients with B cell malignancies. Figure 2  DNA strategy. Nested PCR for immunoglobulin genesusing genomic DNA. (a) Schematic representation of nested-PCR for Figure 3  cDNA strategy. RT-nested PCR for immunoglobulinthe detection of residual tumor cells. Genomic DNA was first ampli-genes using RNA-cDNA samples. (a) Schematic representation of fied using 5 ′  tumor VH family primer and 3 ′  JH primer (VH, JH3), andnested-PCR for the detection of residual tumor cells. Total cDNA wasthen an aliquot of the first reaction was reamplified using inner pri-first amplified using outer primers (CDRII, CH1) and then an aliquotmers derived from tumor CDRIIand CDRIII. Amplified DNA was thenof the first reaction wasreamplified using inner primers (CDRIII,CH2).run on 3% Metaphor gel. (b) PCR dilution experiment demonstratingAmplified DNA was then run on 3% Metaphor gel. (b) PCR dilutionassay sensitivity and specificity in the BJAB cell line. MW, molecularexperiment demonstrating assay sensitivity and specificity in the BJABweight marker; P, BJAB DNA; PL, unrelated polyclonal DNA; N, nocell line. MW, molecular weight marker; P, BJAB DNA; PL, unrelatedDNA control lane.polyclonal cDNA; N, no DNA control lane.  BTS Technical Report C Voena  et al  1797 two different antisense primers from the JH 3 ′  end. This strat- tumor IgH constant region antisense primers are used, increas-ing on one hand the sensitivity and on the other decreasingegy failed to yield satisfactory results: nested-PCR productswere usually very short (50–60 bp) due to deletion of the 5 ′  the risk of false positive results. This procedure always gavecleaner products reducing the possibility of nonspecificend of the JH region and due to few N insertions betweenD segments and JH regions during IgH genes rearrangement. annealing. Moreover, the use of a IgH constant region primeravoids the use of JH antisense primer that sometimes fails toResulting bands were often so faint they were hardlyvisualized on 3% Metaphor gel (data not shown). give amplification because of extensive deletion in itssequence, especially in MM and ALL. 29,30 DNA is a good source for MRD detection when only smearsor old (badly preserved) material are available and RNA Discussion extraction could fail. In this case it is advisable to proceedwith RNA and DNA extraction simultaneously. If RNA extrac-In the present study, we describe a novel nested PCR-methodfor MRD detection in B cell malignancies using the rearrange- tion fails, the DNA strategy provides a possible alternative. Itconsists of an initial amplification with the 5 ′  tumor VH familyment of IgH genes. This method employs tumor-specific pri-mers derived from the tumor CDRs. primer and the 3 ′  JH primer, followed by a second amplifi-cation using CDRII and CDRIII. The use of a VH consensusSeveral methods have been developed to evaluate MRDusing the IgH rearrangement. Most of these methods are based primer in the first PCR may lead to amplification of normalclones unrelated to the tumor one, thus decreasing the sensi-on the amplification of a small portion of the tumor VDJ byusing consensus primers derived from the FR3. Amplification tivity of the second specific amplification.Although the use of the IgH genes as tumor markers stillwith a FR3 primer is often unsuccessful because of the pres-ence of many somatic mutations in the primer template that remains a relatively complex procedure, especially becauseof VDJ sequencing, the use of nested-PCR may greatly simplifyprevent correct annealing rendering this approach of limitedapplication, especially in those B cell malignancies in which B the screening for residual tumor cells in remission samples.This is particularly useful when frequent molecular monitoringlymphocytes have already been exposed to the hypermutationmechanism (MM and NHL). In addition, most of the ALL in is required.which amplification with FR3 primer fail, have a loss of 3 ′ portion of the primer site, presumably as a consequence of exonuclease activity at the time of recombination. 27 More-  Acknowledgements over, such a method allows the reading of the CDRIII only,that is usually 20 nucleotides long (sometimes less than 15) This work has been supported by Associazione Italianaand that can just be employed to derive a tumor-specific Ricerca sul Cancro (AIRC, Milano, Italy) and Consiglioprobe for oligonucleotide hybridization. 11 The FR3 method Nazionale delle Ricerche (Progetto Finalizzato ACROspecificity is then based on one tumor-specific marker and this 9500416.PF39) and BIOMED grant ERBCMRXCT940473. MAmay increase the risk of false positive results. In order to avoid is a recipient of a fellowship from AIRC. We are indebted tothese problems we adopted a different strategy based on the Dr SS Sahota and Dr FK Stevenson for sending us theamplification of a longer fragment of the tumor VDJ by using sequence of framework and leader region families.sense primers derived from the leader or FR1 region. Thisapproach allows the reading of both CDRII and CDRIII. Thereading of two different tumor-specific sequences for each References patient is essential to develop a sensitive and specific methodfor MRD detection characterized by two step specificity: a  1 Vose JM, Armitage JO. Role of autologous bonemarrow transplan-tation in non-Hodgkin lymphoma.  Hematol/Oncol Clin N Am tumor-specific amplification using a CDRII-derived primer 1993;  7 : 577–590. and a tumor-specific hybridization using a CDRIII-derived 2 Gianni AM, Tarella C, Bregni M, Siena S, Lombardi F, Gandola probe. 18,24,28 In order to simplify such an approach, we L, Caracciolo D, Stern A, Bonadonna G, Boccadoro M, Pileri A. devised a nested-PCR method similar to that employed for High-dose sequential chemoradiotherapy, a widely applicable chromosomal translocations. This method maintains two steps  regimen confers survival benefit to patients with high-risk multiplemyeloma.  J Clin Oncol   1994;  12 : 503–509. of specificity since two primers are derived from hypervariable 3 Preti A, Kantarjian HG. Management of adult acute lymphocytic regions of the tumor VDJ, but avoids the use of a hybridization leukemia: present issues and key challenges.  J Clin Oncol   1994; step. Our data show that nested-PCR of the IgH genes is 12 : 1312–1322. widely applicable, simple and fast. Furthermore, since it has 4 Campana D, Pui CH. Detection of minimal residual disease in provided sensitivity and specificity similar to hybridization acute leukemia: methodologic advances and clinical significance. procedures, it could represent a reliable alternative to  Blood   1995;  85 : 1416–1434.5 Gribben JG, Neuberg D, Freedman AS, Gimmi CD, Pesek KW, hybridization methods. Our comparative study to assess Barber M, Saporito L, Woo SD, Coral F, Spector N, Rabinowe SN, whether MRD detection on RNA is better than DNA shows Grossbard ML, Ritz J, Nadler LM. Detection by polymerase chain the advantages of a cDNA strategy. cDNA strategy is more reaction of residual cells with the bcl-2 translocation is associated sensitive than a DNA strategy since, in those specimens in with increased risk of relapse after autologous bone marrow trans- which both methods were applied, amplification of DNA plantation for B-cell lymphoma.  Blood   1993;  81 : 2449–2457. showed a sensitivity usually 10 times lower (10 − 5 vs   10 − 6 ).  6 Brisco MJ,. Condon J, Hughes E, Neoh SH, Sykes PJ, Seshadri R,Toogod I, Waters K, Tauro G, Ekert H, Morley AA. Outcome pre- This result is conceivable since within a cell mRNA is more diction in childhood acute lymphoblastic leukaemia by molecular abundant than DNA. cDNA strategy is, then, a preferable quantification of residual disease at the end of induction.  Lancet  approach as it allows an increase in the yield of the reaction 1994;  343 : 196–200. and possibly enhances the detection of residual tumor cells. 7 Huang W, Sun GL, Li XS, Cao Q, Lu Y, Jang G-S, Zhang F-Q, This strategy is sometimes difficult to achieve because well Chai J-R,Wang ZY, WaxmannS, Chen Z, Chen S-J. Acute promye- preserved samples for RNA extraction are not always avail-  locytic leukemia: clinical relevance of two major PML/RAR-   iso-forms and detection of minimal residual disease by retro- able. In both amplification CDRs, derived sense primers and
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