Association of Clinical Status of Follicular Lymphoma Patients after Autologous Stem Cell Transplant and Quantitative Assessment of Lymphoma in Blood and Bone Marrow as Measured by SYBR Green I Polymerase Chain Reaction
2006; Elsevier BV; Volume: 8; Issue: 1 Linguagem: Inglês
10.2353/jmoldx.2006.050050
ISSN1943-7811
AutoresNancy Pennell, Anthony Woods, Marciano D. Reis, Rena Buckstein, David Spaner, Kevin Imrie, Karen Hewitt, Angela Boudreau, Arun Seth, Neil L. Berinstein,
Tópico(s)T-cell and Retrovirus Studies
ResumoMolecular remission in the autograft and bone marrow after transplant are predictive of durable clinical remission in relapsed follicular lymphoma. Thus, a simple reliable method to quantify minimal residual disease (MRD) would improve prognostication in these patients. Fluorescent hybridization probes have been used in real-time quantitative polymerase chain reaction (RQ-PCR) to monitor MRD with a reproducible sensitivity of 0.01%; however, these techniques are expensive and require additional experiments to examine clonality. We describe a SYBR Green I detection method that is more universal, checks clonal identity, yields the same sensitivity for monitoring MRD, and is more economically attractive. Using this method to follow 14 follicular lymphoma patients treated with autologous stem cell transplantation, molecular markers were successfully defined for 12 patients. Median contamination of stem-cell grafts was 0.1% (range, 0 to 13%). Six patients with measurable graft contamination became PCR-negative in blood and bone marrow within 12 months after autologous stem cell transplantation. Three patients free of disease progression (median follow-up of 75 months) are in molecular remission. Increasing fractions of RQ-PCR-positive blood and bone marrow cells reliably predicted morphological and clinical relapse. In one case, both clinical relapse and spontaneous regression were reflected by changes in MRD levels. Thus, our RQ-PCR method reproducibly distinguishes different levels of MRD. Molecular remission in the autograft and bone marrow after transplant are predictive of durable clinical remission in relapsed follicular lymphoma. Thus, a simple reliable method to quantify minimal residual disease (MRD) would improve prognostication in these patients. Fluorescent hybridization probes have been used in real-time quantitative polymerase chain reaction (RQ-PCR) to monitor MRD with a reproducible sensitivity of 0.01%; however, these techniques are expensive and require additional experiments to examine clonality. We describe a SYBR Green I detection method that is more universal, checks clonal identity, yields the same sensitivity for monitoring MRD, and is more economically attractive. Using this method to follow 14 follicular lymphoma patients treated with autologous stem cell transplantation, molecular markers were successfully defined for 12 patients. Median contamination of stem-cell grafts was 0.1% (range, 0 to 13%). Six patients with measurable graft contamination became PCR-negative in blood and bone marrow within 12 months after autologous stem cell transplantation. Three patients free of disease progression (median follow-up of 75 months) are in molecular remission. Increasing fractions of RQ-PCR-positive blood and bone marrow cells reliably predicted morphological and clinical relapse. In one case, both clinical relapse and spontaneous regression were reflected by changes in MRD levels. Thus, our RQ-PCR method reproducibly distinguishes different levels of MRD. Minimal residual disease (MRD) describes the presence of cancer cells, below the level of detection by standard light microscopy, which is ∼1 in 100 cells. Using polymerase chain reaction (PCR) to assess MRD, the DNA molecular marker from one tumor cell can be detected in a background of DNA of up to 1 million normal nucleated cells. Sensitive PCR testing has been used to examine persistence or recurrence of disease in evaluating new treatment modalities and clearance of autologous stem cell products. Technologies to examine MRD are still evolving, and there is a lack of standard methodology in the tests used in clinical trials. Variability in the sensitivity and precision of the PCR assays can affect the frequency of false-positives and false-negatives and confuse clinical correlations. A quantitative PCR method that is economical and simple to implement with a variety of PCR targets is desirable to replace widely used conventional qualitative PCR.There are currently two genetic features of follicular lymphoma (FL) cells that provide suitable targets for PCR monitoring of residual disease. The first is the t(14;18) (q32;q21) or BCL2/JH translocation characteristic of FL,1Graninger WB Seto M Boutain B Goldman P Korsmeyer SJ Expression of Bcl-2 and Bcl-2-Ig fusion transcripts in normal and neoplastic cells.J Clin Invest. 1987; 80: 1512-1515Crossref PubMed Scopus (245) Google Scholar detectable in nearly all cases by fluorescent in situ hybridization.2Godon A Moreau A Talmant P Baranger-Papot L Genevieve F Milpied N Zandecki M Avet-Loiseau H Is t(14;18)(q32;q21) a constant finding in follicular lymphoma? An interphase FISH study on 63 patients.Leukemia. 2003; 17: 255-259Crossref PubMed Scopus (53) Google Scholar However, the chromosomal breakpoints can occur over a 20,000-bp range. PCR reactions are best suited to amplify small 100- to 500-bp fragments; thus primers that target each breakpoint cluster region should be used to increase the number of patients with PCR-detectable translocations. Sixty percent of BCL2 breakpoints are located at the major breakpoint region (MBR), 5 to 25% at the minor cluster region (mcr) 20,000 bp downstream of MBR, and other breakpoints have been found in clusters between MBR and mcr.3Akasaka T Akasaka H Yonetani N Ohno H Yamabe H Fukuhara S Okuma M Refinement of the BCL2/immunoglobulin heavy chain fusion gene in t(14;18)(q32;q21) by polymerase chain reaction amplification for long targets.Genes Chromosom Cancer. 1998; 21: 17-29Crossref PubMed Scopus (76) Google Scholar, 4Buchonnet G Jardin F Jean N Bertrand P Parmentier F Tison S Lepretre S Contentin N Lenain P Stamatoullas-Bastard A Tilly H Bastard C Distribution of BCL2 breakpoints in follicular lymphoma and correlation with clinical features: specific subtypes or same disease?.Leukemia. 2002; 16: 1852-1856Crossref PubMed Scopus (52) Google Scholar, 5van Dongen JJ Langerak AW Bruggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E Garcia-Sanz R van Krieken JH Droese J Gonzalez D Bastard C White HE Spaargaren M Gonzalez M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2470) Google Scholar Although the BCL2/JH fusion sequence is characteristic of FL, many reports have shown that it is also present in low frequency [∼0.1 to 100 per 1 million cells in peripheral blood (PB)] in normal individuals.6Hirt C Schuler F Dolken G Minimal residual disease (MRD) in follicular lymphoma in the era of immunotherapy with rituximab.Semin Cancer Biol. 2003; 13: 223-231Crossref PubMed Scopus (13) Google Scholar The method used to detect BCL2/JH should distinguish a false-positive result. A second and alternative PCR target for FL is the immunoglobulin heavy chain (IgH) gene rearrangement that is unique to the B-cell clone. In the subset of patients without PCR-detectable BCL2/JH translocation, tumor clones can be identified by PCR amplification of the uniquely rearranged variable-diversity-joining (VDJ) junction in the IgH gene using consensus VH and JH primers.7von Neuhoff N Dreger P Suttorp M Marget M Kell S Schmitz N Comparison of different strategies of molecular genetic monitoring following autologous stem cell transplantation in patients with follicular lymphoma.Bone Marrow Transplant. 1998; 22: 161-166Crossref PubMed Scopus (19) Google Scholar The unique VDJ product can then be sequenced to identify rearrangements by use of allele-specific oligonucleotide (ASO) PCR primers.Although qualitative analysis of BCL2/JH with sensitive nested PCR8Gribben JG Freedman A Woo SD Blake K Shu RS Freeman G Longtine JA Pinkus GS Nadler LM All advanced stage non-Hodgkin's lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangement at evaluation and after treatment.Blood. 1991; 78: 3275-3280PubMed Google Scholar has been widely used, newer methods of quantification of MRD can be more informative. Several real-time quantitative PCR technologies are available and have recently been reviewed.9van der Velden VH Hochhaus A Cazzaniga G Szczepanski T Gabert J van Dongen JJ Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects.Leukemia. 2003; 17: 1013-1034Crossref PubMed Scopus (441) Google Scholar Fundamentally, fluorescent probes or PCR products are used to track the minimum number of PCR cycles required to generate measurable threshold amounts of PCR product as the reaction proceeds (real-time). The number of cycles to reach threshold is inversely related to the number of target templates in a sample.Fluorescently labeled probes generate signal through hybridization of target PCR products and subsequent interaction with Taq polymerase. In these methodologies, each PCR amplification primer set has an associated specific probe oligonucleotide, which adds additional challenge to the design of the PCR reaction. Labeled probes are expensive to synthesize, so to be able to do quantitative assessments of a range of different breakpoint regions, a number of costly specific probes are required. Additionally, product analysis after PCR on agarose gels, capillary electrophoresis,10Sanchez-Vega B Vega F Medeiros LJ Lee MS Luthra R Quantification of bcl-2/JH fusion sequences and a control gene by multiplex real-time PCR coupled with automated amplicon sizing by capillary electrophoresis.J Mol Diagn. 2002; 4: 223-229Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar or by DNA sequencing is required to examine clonality. Methods have been developed for monitoring BCL2/JH with dual-labeled TaqMan (Applied Biosystems, Foster City, CA) hydrolysis probes for the BCL2 gene.10Sanchez-Vega B Vega F Medeiros LJ Lee MS Luthra R Quantification of bcl-2/JH fusion sequences and a control gene by multiplex real-time PCR coupled with automated amplicon sizing by capillary electrophoresis.J Mol Diagn. 2002; 4: 223-229Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 11Luthra R McBride JA Cabanillas F Sarris A Novel 5′ exonuclease-based real-time PCR assay for the detection of t(14;18)(q32;q21) in patients with follicular lymphoma.Am J Pathol. 1998; 153: 63-68Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 12Olsson K Gerard CJ Zehnder J Jones C Ramanathan R Reading C Hanania EG Real-time t(11;14) and t(14;18) PCR assays provide sensitive and quantitative assessments of minimal residual disease (MRD).Leukemia. 1999; 13: 1833-1842Crossref PubMed Scopus (51) Google Scholar, 13Ladetto M Sametti S Donovan JW Ferrero D Astolfi M Mitterer M Ricca I Drandi D Corradini P Coser P Pileri A Gribben JG Tarella C A validated real-time quantitative PCR approach shows a correlation between tumor burden and successful ex vivo purging in follicular lymphoma patients.Exp Hematol. 2001; 29: 183-193Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 14Hirt C Dolken G Quantitative detection of t(14;18)-positive cells in patients with follicular lymphoma before and after autologous bone marrow transplantation.Bone Marrow Transplant. 2000; 25: 419-426Crossref PubMed Scopus (48) Google Scholar Clinical studies incorporating these BCL2 probes14Hirt C Dolken G Quantitative detection of t(14;18)-positive cells in patients with follicular lymphoma before and after autologous bone marrow transplantation.Bone Marrow Transplant. 2000; 25: 419-426Crossref PubMed Scopus (48) Google Scholar, 15Ghielmini M Schmitz SF Cogliatti SB Pichert G Hummerjohann J Waltzer U Fey MF Betticher DC Martinelli G Peccatori F Hess U Zucca E Stupp R Kovacsovics T Helg C Lohri A Bargetzi M Vorobiof D Cerny T Prolonged treatment with rituximab in patients with follicular lymphoma significantly increases event-free survival and response duration compared with the standard weekly x 4 schedule.Blood. 2004; 103: 4416-4423Crossref PubMed Scopus (531) Google Scholar limited analysis to patients with MBR breakpoints, thus possibly excluding results from up to 40% of study patients. A diagnostic kit that utilizes labeled hybridization probes to detect BCL2/JH translocations is available, but it is restricted for use on MBR region breakpoints (Roche Applied Science (Mannheim, Germany) catalog no. 3062651). A method using probes for the JH gene16Jenner MJ Summers KE Norton AJ Amess JA Arch RS Young BD Lister TA Fitzgibbon J Goff LK JH probe real-time quantitative polymerase chain reaction assay for Bcl-2/IgH rearrangements.Br J Haematol. 2002; 118: 550-558Crossref PubMed Scopus (10) Google Scholar required sequencing each patient BCL2/JH PCR product to determine the correct probe to use for quantitative PCR analysis. Although RQ-PCR using IgH-ASO strategies with consensus VH probes have been used for acute lymphoblastic leukemia patients,17Donovan JW Ladetto M Zou G Neuberg D Poor C Bowers D Gribben JG Immunoglobulin heavy-chain consensus probes for real-time PCR quantification of residual disease in acute lymphoblastic leukemia.Blood. 2000; 95: 2651-2658PubMed Google Scholar due to the frequency of VH region somatic mutations in FL it has been more challenging to develop consensus probes to detect ASO-IgH in FL patients.18Yashima A Maesawa C Uchiyama M Tarusawa M Satoh T Satoh M Enomoto S Sugawara K Numaoka H Murai K Utsugisawa T Ishida Y Masuda T Quantitative assessment of contaminating tumor cells in autologous peripheral blood stem cells of B-cell non-Hodgkin lymphomas using immunoglobulin heavy chain gene allele-specific oligonucleotide real-time quantitative-polymerase chain reaction.Leuk Res. 2003; 27: 925-934Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar It is both expensive and time consuming to use patient-specific probes in a clinical study involving large numbers of patients.Alternatively, the nonspecific fluorescent intercalating dye SYBR Green I can be used. SYBR Green I becomes excited when it binds to the double-stranded DNA amplicon, and the recorded fluorescence is a direct measurement of PCR product quantity. Additionally, the melting temperature of the fluorescent amplicon can be used to examine clonality in the same run. SYBR Green I is less costly because no labeled sequence-specific probes are required. A wide range of existing PCR reactions can readily be adapted to the SYBR Green I assay and monitored with the same reagents. Real-time PCR instruments with single color detection are generally less expensive than multicolor detection systems. SYBR Green I has been used to detect t(14;18) MBR translocations,19Galimberti S Guerrini F Morabito F Palumbo GA Di Raimondo F Papineschi F Caracciolo F Fazzi R Cervetti G Cuzzocrea A Petrini M Quantitative molecular evaluation in autotransplant programs for follicular lymphoma: efficacy of in vivo purging by Rituximab.Bone Marrow Transplant. 2003; 32: 57-63Crossref PubMed Scopus (41) Google Scholar, 20Bohling SD King TC Wittwer CT Elenitoba-Johnson KS Rapid simultaneous amplification and detection of the MBR/JH chromosomal translocation by fluorescence melting curve analysis.Am J Pathol. 1999; 154: 97-103Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar but these studies did not examine the value of these assays in long-term follow-up serial monitoring. In small trials, it can be essential to obtain molecular data on as many patients as possible in an economical way. In this study, we have implemented a SYBR Green I approach to detect t(14;18) with four different sets of primers and patient-specific clonal IgH enabling us to monitor the disease status of most of our patients. We compared previously generated qualitative nested PCR data to RQ-PCR for monitoring MRD in a clinical study of FL patients receiving autologous stem cell transplantation (ASCT) followed by maintenance treatment with interferon-α.Materials and MethodsPatientsAdult patients aged 18 to 65 years with good performance status and less than or equal to three relapses of FL grade 1 or 2, according to the World Health Organization Classification21Harris NL Jaffe ES Diebold J Flandrin G Muller-Hermelink HK Vardiman J Lister TA Bloomfield CD 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 (2499) Google Scholar of lymphoma, were treated in this noncomparative, prospective, nonrandomized phase II clinical trial. All patients meeting eligibility requirements were included (Table 1). Patients were treated at the Toronto-Sunnybrook Regional Cancer Centre. The hospital institutional review board and ethics committees approved the trial, and informed consent was obtained for all patients. Patients received initial debulking chemotherapy with standard cyclophosphamide, doxorubicin, vincristine, prednisone or dexamethasone, cisplatin, and cytarabine in the event of previous anthracycline exposure. Stem-cell collection took place when bone marrow (BM) involvement with lymphoma was <15% as determined by histology. Stem cells were mobilized with 5 days of high-dose granulocyte colony-stimulating factor. Patients achieving at least a 75% reduction in tumor bulk proceeded to high-dose therapy with standard cyclophosphamide, carmustine, and etoposide conditioning and ASCT. Immunotherapy after transplant consisted of subcutaneous interferon-α 3 million U/m2 three times weekly beginning 3 months after ASCT for a period of 24 months. Response to therapy was assessed by serial clinical examination, blood counts and chemistries, BM aspiration and biopsy, and computed tomography (CT) scans of involved sites. These evaluations took place 8 weeks after ASCT, then every 3 months for the first year after ASCT, and every 6 months thereafter.Table 1Patient CharacteristicsCategoryScaleNumberAge (years)Median48Range35–56SexFemale4Male10International PrognosticLow7Index (IPI) scoreLow intermediate6High intermediate1Prior therapiesMedian1Range1–3Current status (months)Median follow-up41Range9–75Median disease-free survival42Median overall survivalNot reached Open table in a new tab Sample CollectionBaseline samples of lymph node (LN), BM, or PB assessed as morphologically positive for disease involvement were analyzed by PCR as described below, for the presence of molecular markers for t(14;18) translocations.22Iqbal S Jenner MJ Summers KE Davies AJ Matthews J Norton AJ Calaminici M Rohatiner AZ Fitzgibbon J Lister TA Goff LK Reliable detection of clonal IgH/Bcl2 MBR rearrangement in follicular lymphoma: methodology and clinical significance.Br J Haematol. 2004; 124: 325-328Crossref PubMed Scopus (16) Google Scholar If t(14;18) was not detectable, a minimum of 1% disease infiltration was desirable to identify a clonal IgH marker. The determination of baseline infiltration of the BM sample was achieved by combined morphological and immunohistochemical analysis, the latter using antibodies to CD20 and bcl-2. Quantities are expressed as a percentage of the visual stained lymphocytes. For patients in whom a baseline molecular marker was identified, serial molecular monitoring for MRD was performed on all BM and PB samples (before apheresis, before transplant, and after ASCT at specified 3- to 6-month follow-up intervals). The stem cell graft was similarly evaluated for the presence of occult disease.DNA PreparationsDNA was extracted from fresh or frozen LN tissue, buffy-coated BM aspirates, or buffy-coated PB using Qiagen DNA mini kit (Qiagen, Valencia, CA). BM biopsy specimens were found to be unreliable for PCR analysis as yields were low and fixation and decalcification processes often degraded the DNA. Therefore, only BM aspirates were analyzed in follow-up samples. DNA concentrations were adjusted to be in the range of 80 to 140 ng/μL. The translation from DNA concentration to cell number is dependant on the total amount of amplifiable DNA in the sample. We have generated primers to amplify a 350-bp fragment of a highly conserved gene, RAG2,23Zarrin AA Fong I Malkin L Marsden PA Berinstein NL Cloning and characterization of the human recombination activating gene 1 (RAG1) and RAG2 promoter regions.J Immunol. 1997; 159: 4382-4394PubMed Google Scholar as a control for both DNA quality and quantity.Nested PCR ReactionsPrecautions were taken to separate pre- and post-PCR work areas and to prevent potential contamination by PCR products. Negative controls included DNA from Jur-kat T-cell lymphoma cell line, which lacked the B-cell-specific translocations. For a positive control of MBR translocations, OCI LY8 cell line24Mehra S Messner H Minden M Chaganti RS Molecular cytogenetic characterization of non-Hodgkin lymphoma cell lines.Genes Chromosom Cancer. 2002; 33: 225-234Crossref PubMed Scopus (55) Google Scholar was serially diluted in Jurkat DNA. The minor cluster region (mcr)-positive control was patient LN DNA.Nested PCR for t(14;18) followed by gel electrophoresis was performed with primers for the MBR and mcr of the BCL2 gene, and JH of the immunoglobulin heavy chain gene as described previously.8Gribben JG Freedman A Woo SD Blake K Shu RS Freeman G Longtine JA Pinkus GS Nadler LM All advanced stage non-Hodgkin's lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangement at evaluation and after treatment.Blood. 1991; 78: 3275-3280PubMed Google Scholar Between 500 ng and 1 μg of patient sample was amplified with 0.5 U of Hot Star Taq polymerase (Qiagen), external MBR (or mcr)-JH primers and multiplexed with RAG2 primers (Table 2) in a 50-μl reaction volume. The reaction began with a 15-minute incubation at 94°C to activate the enzyme, followed by 25 cycles (1 minute at 94°C, 1 minute at 55°C, and 1 minute at 72°C). Subsequently, 5 μl of the PCR product was reamplified with internal MBR (or mcr)-JH primers for 30 cycles with an annealing temperature of 58°C. PCR products were analyzed on ethidium bromide-stained 2% agarose gels.Table 2Primers for t(14;18) Translocation Detection and the RAG2 Gene ControlPrimerSequenceReferenceMBR (nested external)5′-CAGCCTTGAAACATTGATGG-3′8Gribben JG Freedman A Woo SD Blake K Shu RS Freeman G Longtine JA Pinkus GS Nadler LM All advanced stage non-Hodgkin's lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangement at evaluation and after treatment.Blood. 1991; 78: 3275-3280PubMed Google ScholarMBR (nested internal)5′-TATGGTGGTTTGACCTTTAG-3′8Gribben JG Freedman A Woo SD Blake K Shu RS Freeman G Longtine JA Pinkus GS Nadler LM All advanced stage non-Hodgkin's lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangement at evaluation and after treatment.Blood. 1991; 78: 3275-3280PubMed Google Scholarmcr (nested external)5′-CGTGCTGGTACCACTCCTG-3′8Gribben JG Freedman A Woo SD Blake K Shu RS Freeman G Longtine JA Pinkus GS Nadler LM All advanced stage non-Hodgkin's lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangement at evaluation and after treatment.Blood. 1991; 78: 3275-3280PubMed Google Scholarmcr (nested internal)5′-GGACCTTCCTTGGTGTGTTG-3′8Gribben JG Freedman A Woo SD Blake K Shu RS Freeman G Longtine JA Pinkus GS Nadler LM All advanced stage non-Hodgkin's lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangement at evaluation and after treatment.Blood. 1991; 78: 3275-3280PubMed Google ScholarMBR E5′-TCCAGATGGCGAATGACCAG-3′MBR B5′-GAGTTGCTTTACGTGG-3′20Bohling SD King TC Wittwer CT Elenitoba-Johnson KS Rapid simultaneous amplification and detection of the MBR/JH chromosomal translocation by fluorescence melting curve analysis.Am J Pathol. 1999; 154: 97-103Abstract Full Text Full Text PDF PubMed Scopus (23) Google ScholarMBR H5′-GCAACAGAGAACCATCCCT-3′26Coad JE Olson DJ Christensen DR Lander TA Chibbar R McGlennen RC Brunning RD Correlation of PCR-detected clonal gene rearrangements with bone marrow morphology in patients with B-lineage lymphomas.Am J Surg Pathol. 1997; 21: 1047-1056Crossref PubMed Scopus (55) Google ScholarJH5′-ACCTGAGGAGACGGTGACC-3′4Buchonnet G Jardin F Jean N Bertrand P Parmentier F Tison S Lepretre S Contentin N Lenain P Stamatoullas-Bastard A Tilly H Bastard C Distribution of BCL2 breakpoints in follicular lymphoma and correlation with clinical features: specific subtypes or same disease?.Leukemia. 2002; 16: 1852-1856Crossref PubMed Scopus (52) Google ScholarMcr5′-CGTGCTGGTACCACTCCTG-3′4Buchonnet G Jardin F Jean N Bertrand P Parmentier F Tison S Lepretre S Contentin N Lenain P Stamatoullas-Bastard A Tilly H Bastard C Distribution of BCL2 breakpoints in follicular lymphoma and correlation with clinical features: specific subtypes or same disease?.Leukemia. 2002; 16: 1852-1856Crossref PubMed Scopus (52) Google ScholarRAG 2 Forward (F1)5′-CCTGAAGCCAGATATGGTC-3′23Zarrin AA Fong I Malkin L Marsden PA Berinstein NL Cloning and characterization of the human recombination activating gene 1 (RAG1) and RAG2 promoter regions.J Immunol. 1997; 159: 4382-4394PubMed Google ScholarRAG 2 Reverse (R1)5′-GTCCAATTCACAGCTGGGCT-3′23Zarrin AA Fong I Malkin L Marsden PA Berinstein NL Cloning and characterization of the human recombination activating gene 1 (RAG1) and RAG2 promoter regions.J Immunol. 1997; 159: 4382-4394PubMed Google Scholar Open table in a new tab In patients for whom t(14;18) was not detectable, an attempt was made to determine clonal-specific IgH primers using the method described by Andersen and colleagues.25Andersen NS Donovan JW Borus JS Poor CM Neuberg D Aster JC Nadler LM Freedman AS Gribben JG Failure of immunologic purging in mantle cell lymphoma assessed by polymerase chain reaction detection of minimal residual disease.Blood. 1997; 90: 4212-4221PubMed Google Scholar Essentially, VH framework I family-specific and JH consensus primers were used to amplify the clonal IgH fragment. This DNA was then sequenced and analyzed with IgBLAST, a software program specifically designed for comparing IgH DNA sequences available from the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov). From this analysis, patient allele-specific primers were designed with the sense primer located in VH framework 1 region and the anti-sense primer from the CDR3 region (Figure 1).RQ-PCR ReactionsAmplification reactions were performed using QuantiTect SYBR Green PCR kit (catalog no. 204143, Qiagen) with each reaction containing 500 ng of sample DNA and 0.2 μmol/L to 0.3 μmol/L primers in a 50-μl volume. This kit was optimal for PCR products between 100 and 600 bp, so new primers for MBR were incorporated into the RQ-PCR protocols to amplify patient samples whose MBR-JH PCR product was larger than 500-bp as determined by agarose gel analysis. RQ-PCR primers, cycle conditions, and positive controls are listed in Table 2, Table 3 and mapped in Figure 1. Primers for each patient sample were selected based on previously generated positive results from qualitative PCR analysis. A 500-ng sample of patient DNA represents 8 × 104 cells. Patient samples were amplified with RAG2 primers in a separate reaction to determine cell equivalents. The standard curve for this reaction was derived with 10-fold serial dilutions of DNA from Jurkat cells. Quantification of the MBR/JH for MBRE and MBRB primer sets was determined from a standard curve established with 10-fold serial dilutions of DNA from OCI LY8 diluted into BCL2/JH-negative Jurkat DNA. The positive control standard curves for MBRH, mcr, and ASO PCR reactions were generated using DNA from highly infiltrated patient LN DNA.Table 3Real-Time PCR Reaction ConditionsCycle conditionsReactionPrimersAnnealing (°C)No. of cyclesPositive controls1MBR E + JH6345Ly8 cell line [23]2MBR B + JH6345Ly8 cell line3MBR H + JH5845Pt lymph node DNA4mcr + JH6037Pt lymph node DNA5RAG2 F1 + RAG2 R15845Jurkat's cell lineThe reactions began with a 15-minute incubation at 95°C to activate HotStarTaq DNA polymerase. This was followed by the indicated number of cycles of 20 seconds at 92°C, 20 seconds at the appropriate annealing temperature, 20 seconds at 72°C, followed by plate read. The melt curve was performed at the end of the amplification reactions; from 65.0 to 95.0°C; read every 0.2°C; hold for 0.01 seconds between reads. Open table in a new tab Thermocycling was conducted using a DNA Engine Opticon (MJ Research, Boston, MA) initiated by a 15-minute incubation at 94°C followed by cycles and annealing temperatures determined by each primer set as detailed in Table 3. A plate read was performed at each cycle after the 72°C extension. Each run was completed with a melting curve analysis to confirm specificity of amplification and also to verify clonal identity where possible. Cycle threshold (Ct) values were determined by the Opticon software using a fluorescence threshold manually set to 0.02 for all runs. Data were exported to Microsoft Excel for further analysis. For both nested and RQ-PCR, molecular markers were confirmed by testing more than one tissue or sample time point. All follow-up samples were analyzed under the same conditions accompanied by baseline samples as additional positive controls. All patient samples were analyzed in triplicate.ResultsMolecular MarkersMol
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