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• W2005706794 abstract "Translocation t(9;22), which produces the BCR-ABL gene, is pathognomonic of chronic myeloid leukemia. For clinical purposes, the amount of chimeric transcript is considered proportional to the leukemic clone; thus, mRNA is commonly used for molecular monitoring of patients. However, there is no consensus regarding the degree of increase in mRNA that should cause concern or whether the absence of transcript indicates a “cure.” In this study, we analyzed 57 samples from 10 chronic myeloid leukemia patients undergoing imatinib treatment. For each sample, we compared BCR-ABL mRNA levels with the actual proportion of leukemic cells, which were measured through a novel genomic approach based on the quantitative amplification of DNA breakpoints. The two approaches gave similar patterns of residual disease, and the majority of patients were still positive after an average treatment period of 2 years. Nevertheless, in one of two patients with confirmed undetectable levels of chimeric transcript, DNA still revealed the persistence of leukemic cells at 42 months. These findings appear to justify the clinical practice of maintaining imatinib treatment indefinitely. However, the absence of leukemic DNA (observed in 1 of 10 patients) could be used to identify possible candidates for drug discontinuation. In conclusion, DNA analysis proved to be a reliable index of residual disease with potential applications in the field of clinical diagnostics and research. Translocation t(9;22), which produces the BCR-ABL gene, is pathognomonic of chronic myeloid leukemia. For clinical purposes, the amount of chimeric transcript is considered proportional to the leukemic clone; thus, mRNA is commonly used for molecular monitoring of patients. However, there is no consensus regarding the degree of increase in mRNA that should cause concern or whether the absence of transcript indicates a “cure.” In this study, we analyzed 57 samples from 10 chronic myeloid leukemia patients undergoing imatinib treatment. For each sample, we compared BCR-ABL mRNA levels with the actual proportion of leukemic cells, which were measured through a novel genomic approach based on the quantitative amplification of DNA breakpoints. The two approaches gave similar patterns of residual disease, and the majority of patients were still positive after an average treatment period of 2 years. Nevertheless, in one of two patients with confirmed undetectable levels of chimeric transcript, DNA still revealed the persistence of leukemic cells at 42 months. These findings appear to justify the clinical practice of maintaining imatinib treatment indefinitely. However, the absence of leukemic DNA (observed in 1 of 10 patients) could be used to identify possible candidates for drug discontinuation. In conclusion, DNA analysis proved to be a reliable index of residual disease with potential applications in the field of clinical diagnostics and research. Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the reciprocal translocation t(9;22)(q34;q11). Consequences of the translocation are the Philadelphia (Ph) chromosome and its molecular counterpart, the BCR-ABL fusion gene, which encodes for a deregulated tyrosine kinase. In the absence of intervention, CML evolves from an initial chronic phase (CP) to an accelerated phase and ultimately to a blast crisis.1Melo JV Barnes DJ Chronic myeloid leukaemia as a model of disease evolution in human cancer.Nat Rev Cancer. 2007; 7: 441-453Crossref PubMed Scopus (479) Google Scholar First-line therapy for patients in the CP is based on the tyrosine kinase inhibitor imatinib mesylate (IM) (Novartis Pharmaceuticals, Basel, Switzerland), which prolongs event-free and overall survival2Hughes TP Kaeda J Branford S Rudzki Z Hochhaus A Hensley ML Gathmann I Bolton AE van Hoomissen IC Goldman JM Radich JP Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia.N Engl J Med. 2003; 349: 1423-1432Crossref PubMed Scopus (1064) Google Scholar but does not seem to eradicate the malignancy.3Elrick LJ Jorgensen HG Mountford JC Holyoake TL Punish the parent not the progeny.Blood. 2005; 105: 1862-1866Crossref PubMed Scopus (132) Google Scholar Before the advent of IM, allogeneic stem cell transplantation was a common therapy and still remains an option. This procedure is associated with an appreciable level of morbidity and mortality but increases the probability of long-term leukemia-free survival.4Mughal TI Yong A Szydlo RM Dazzi F Olavarria E van Rhee F Kaeda J Cross NC Craddock C Kanfer E Apperley J Goldman JM Molecular studies in patients with chronic myeloid leukaemia in remission 5 years after allogeneic stem cell transplant define the risk of subsequent relapse.Br J Haematol. 2001; 115: 569-574Crossref PubMed Scopus (58) Google Scholar In contrast, most patients who stop taking IM quickly lose the response they achieved.5Cortes J O'Brien S Kantarjian H Discontinuation of imatinib therapy after achieving a molecular response.Blood. 2004; 104: 2204-2205Crossref PubMed Scopus (254) Google Scholar,6Usuki K Iijima K Iki S Urabe A CML cytogenetic relapse after cessation of imatinib therapy.Leuk Res. 2005; 29: 237-238Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar As a consequence, IM therapy is indefinitely prolonged and patients are monitored regularly. Thus, decisions about the clinical management of CML should be informed by adverse effects7Deininger MW O'Brien SG Ford JM Druker BJ Practical management of patients with chronic myeloid leukemia receiving imatinib.J Clin Oncol. 2003; 21: 1637-1647Crossref PubMed Scopus (312) Google Scholar and the costs8Goldman J Is imatinib a cost-effective treatment for newly diagnosed chronic myeloid leukemia patients?.Nat Clin Practice Oncol. 2005; 2: 126-127Crossref PubMed Scopus (4) Google Scholar of continuous treatment. Response to IM can be assessed over time by observing the reduction of leukocytes, Ph-positive cells, and BCR-ABL transcripts.9Goldman J Monitoring minimal residual disease in BCR-ABL-positive chronic myeloid leukemia in the imatinib era.Curr Opin Hematol. 2005; 12: 33-39Crossref PubMed Scopus (53) Google Scholar A detailed definition of hematological, cytogenetic, and molecular responses is closely connected to treatment failure and a suboptimal outcome.10Baccarani M Saglio G Goldman J Hochhaus A Simonsson B Appelbaum F Apperley J Cervantes F Cortes J Deininger M Gratwohl A Guilhot F Horowitz M Hughes T Kantarjian H Larson R Niederwieser D Silver R Hehlmann R Evolving concepts in the management of chronic myeloid leukemia: recommendations from an expert panel on behalf of the European LeukemiaNet.Blood. 2006; 108: 1809-1820Crossref PubMed Scopus (1074) Google Scholar Complete cytogenetic response has been defined as the absence of Ph+ cells and can be achieved in 75% to 90% of patients treated in the early CP.11Kantarjian H Talpaz M O'Brien S Garcia-Manero G Verstovsek S Giles F Rios MB Shan J Letvak L Thomas D Faderl S Ferrajoli A Cortes J High-dose imatinib mesylate therapy in newly diagnosed Philadelphia chromosome-positive chronic phase chronic myeloid leukemia.Blood. 2004; 103: 2873-2878Crossref PubMed Scopus (354) Google Scholar For these patients residual leukemia can be assessed only by reverse-transcription quantitative PCR (RQ-PCR). The detection of BCR-ABL mRNA reflects the presence of leukemic cells in a semiquantitative manner, and a rising level is considered an early indication of failing response. However, there is still no consensus regarding the degree of the increase in mRNA that should cause concern.12Branford S Rudzki Z Parkinson I Grigg A Taylor K Seymour JF Durrant S Browett P Schwarer AP Arthur C Catalano J Leahy MF Filshie R Bradstock K Herrmann R Joske D Lynch K Hughes T Realtime quantitative PCR analysis can be used as a primary screen to identify patients with CML treated with imatinib who have BCR-ABL kinase domain mutations.Blood. 2004; 104: 2926-2932Crossref PubMed Scopus (199) Google Scholar,13Wang L Knight K Lucas C Clark RE The role of serial BCR-ABL transcript monitoring in predicting the emergence of BCR-ABL kinase mutations in imatinib-treated patients with chronic myeloid leukemia.Haematologica. 2006; 91: 235-239PubMed Google Scholar This problem is hampered by the observation that apparent levels of chimeric transcript may be detected in healthy persons14Bose S Deininger M Gora-Tybor J Goldman JM Melo JV The presence of typical and atypical BCR-ABL fusion genes in leukocytes of normal individuals: biologic significance and implications for the assessment of minimal residual disease.Blood. 1998; 92: 3362-3367PubMed Google Scholar,15Biernaux C Loos M Sels A Huez G Stryckmans P Detection of major bcr-abl gene expression at a very low level in blood cells of some healthy individuals.Blood. 1995; 86: 3118-3122PubMed Google Scholar resulting in a background noise. In 2003, the Europe against Cancer (EAC) network established a standardized protocol for the quantitative detection of BCR-ABL transcripts.16Gabert J Beillard E van der Velden VH Bi W Grimwade D Pallisgaard N Barbany G Cazzaniga G Cayuela JM Cavé H Pane F Aerts JL De Micheli D Thirion X Pradel V González M Viehmann S Malec M Saglio G van Dongen JJ Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia—a Europe Against Cancer program.Leukemia. 2003; 17: 2318-2357Crossref PubMed Scopus (1213) Google Scholar Currently there are various methods for reporting results, one of which is to normalize the amount BCR-ABL mRNA against an appropriate control transcript. More recently, expression levels were reported on a log10 reduction scale from a standardized baseline for untreated patients.2Hughes TP Kaeda J Branford S Rudzki Z Hochhaus A Hensley ML Gathmann I Bolton AE van Hoomissen IC Goldman JM Radich JP Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia.N Engl J Med. 2003; 349: 1423-1432Crossref PubMed Scopus (1064) Google Scholar The use of this scale has led to some degree of confusion, because it seems to imply that the value is a relative one. For this reason, a standardized international scale has been recently introduced,17Hughes T Deininger M Hochhaus A Branford S Radich J Kaeda J Baccarani M Cortes J Cross NC Druker BJ Gabert J Grimwade D Hehlmann R Kamel-Reid S Lipton JH Longtine J Martinelli G Saglio G Soverini S Stock W Goldman JM Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for “harmonizing” current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results.Blood. 2006; 108: 28-37Crossref PubMed Scopus (994) Google Scholar but its use is still limited, since the required reference materials are difficult to access. RNA technology is also affected by other limitations. Different RQ-PCR assays have been developed,17Hughes T Deininger M Hochhaus A Branford S Radich J Kaeda J Baccarani M Cortes J Cross NC Druker BJ Gabert J Grimwade D Hehlmann R Kamel-Reid S Lipton JH Longtine J Martinelli G Saglio G Soverini S Stock W Goldman JM Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for “harmonizing” current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results.Blood. 2006; 108: 28-37Crossref PubMed Scopus (994) Google Scholar and a number of different control genes (mainly BCR, ABL, and GUSB) are in use.18Beillard E Pallisgaard N van der Velden VH Bi W Dee R van der Schoot E Delabesse E Macintyre E Gottardi E Saglio G Watzinger F Lion T van Dongen JJ Hokland P Gabert J Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using ‘real-time’ quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR)—a Europe against cancer program.Leukemia. 2003; 17: 2474-2486Crossref PubMed Scopus (718) Google Scholar Many of these protocols are far from being exhaustively tested, and expression levels are further influenced by the RNA extraction and the reverse transcription procedures.19Müller MC Hördt T Paschka P Merx K La Rosée P Hehlmann R Hochhaus A A standardization of preanalytical factors for minimal residual disease analysis in chronic myelogenous leukemia.Acta Haematol. 2004; 112: 30-33Crossref PubMed Scopus (28) Google Scholar The inability to eradicate the disease through pharmacological treatment is probably due to the persistence of primitive hematopoietic progenitors3Elrick LJ Jorgensen HG Mountford JC Holyoake TL Punish the parent not the progeny.Blood. 2005; 105: 1862-1866Crossref PubMed Scopus (132) Google Scholar that are highly resistant to IM.20Graham SM Jørgensen HG Allan E Pearson C Alcorn MJ Richmond L Holyoake TL Primitive, quiescent Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro.Blood. 2002; 99: 319-325Crossref PubMed Scopus (987) Google Scholar In the last few years, a great effort was expended in an attempt to classify the leukemic “stem” cells in CML,21Kavalerchik E Goff D Jamieson CH Chronic myeloid leukemia stem cells.J Clin Oncol. 2008; 10: 2911-2915Crossref Scopus (88) Google Scholar,22Savona M Talpaz M Getting to the stem of chronic myeloid leukaemia.Nat Rev Cancer. 2008; 8: 341-350Crossref PubMed Scopus (147) Google Scholar but an accurate characterization is still lacking. In particular, it is not clear if these cells are transcriptionally silent,23Goldman J Gordon M Why do chronic myelogenous leukemia stem cells survive allogeneic stem cell transplantation or imatinib: does it really matter?.Leukemia Lymphoma. 2006; 47: 1-7Crossref PubMed Scopus (104) Google Scholar or not.24Barnes DJ Melo VJ Primitive, quiescent and difficult to kill: the role of non-proliferating stem cells in chronic myeloid leukemia.Cell Cycle. 2006; 5: 2862-2866Crossref PubMed Scopus (111) Google Scholar This is of relevance, since molecular monitoring of CML patients is a circumstantial process based on the quantification of mRNA, rather than a direct observation of the leukemic clone. For these reasons, a definition of complete molecular response is still lacking, and whether the absence of chimeric transcript is consonant with “cure” remains an open question. To better investigate the relationship between the proportion of leukemic cells and the expression of the BCR-ABL fusion gene, we developed a DNA-based assay to be used in parallel with conventional RQ-PCR to monitor patients with CML. A similar study has been previously undertaken only by Zhang et al25Zhang JG Lin F Chase A Goldman JM Cross NC Comparison of genomic DNA and cDNA for detection of residual disease after treatment of chronic myeloid leukemia with allogeneic bone marrow transplantation.Blood. 1996; 87: 2588-2593PubMed Google Scholar in 1996. In that report mRNA from 10 patients after bone marrow transplantation was analyzed by competitive RT-PCR, while the presence of leukemic cells was assessed by nested PCR on genomic DNA. The majority of samples (79%) gave concordant results, thus discrepancies were attributed only to the different sensitivity between competitive RT-PCR and nested PCR. The authors concluded that patients do not generally have a pool of transcriptionally silent cells and mRNA analysis is a full satisfactory procedure for molecular monitoring of residual disease. However, recent improvements to quantitative PCR and the introduction of IM as front-line treatment revealed a new interest in these arguments. In this paper, we reported evidence of how the analysis of DNA from CML patients by real-time PCR could help the research and improve the practice of conventional molecular diagnostics. We monitored, for an average period of 25.5 months (range, 18–42), 10 patients with CML diagnosed in the early CP. All participants gave written informed consent and the research protocol was approved by the Ethic Committee of the Università dell'Insubria. Genomic junctions of BCR-ABL had been previously characterized in Mattarucchi et al.26Mattarucchi E Guerini V Rambaldi A Campiotti L Venco A Pasquali F Lo Curto F Porta G Microhomologies and interspersed repeat elements at genomic breakpoints in chronic myeloid leukemia.Genes Chromosomes Cancer. 2008; 47: 625-632Crossref PubMed Scopus (41) Google Scholar In the same paper, a detailed explanation of the sequencing protocol was also reported. Briefly, DNA from patients designated as 1, 2, 3, 4, 6, 8, 9, 10, PR1, and PR2 was extracted from blood or bone marrow, fragmented, ligated to adaptors, and amplified by a nested PCR using a BCR specific forward primer. Thus, genomic breakpoints were sequenced. The majority of patients were treated with IM monotherapy at a starting dose of 400 mg/day, except for patient 9 who was participating in an 800-mg/day trial. The dose for patients 6 and 10 was increased to 600 mg/day, as a consequence of suboptimal cytogenetic findings observed at 12 and 6 months, respectively. Conventional laboratory investigations (both cytogenetics and mRNA analysis) were performed at the reference laboratory of the CML network of the Italian Group of Hematological Malignancies in Adults at the Hospital of Bergamo, Italy. All of the analytical procedures were subject to the quality control process according to the ISO 9001:2000 accreditation of the laboratory and samples that did not conform were rejected. A cytogenetic analysis of bone marrow (approximately 20 metaphases for each patient) was performed before treatment using conventional QFQ staining. Cytogenetic tests were repeated every 6 months, until a complete response was achieved. Then bone marrow metaphases were analyzed less frequently. Levels of BCR-ABL mRNA were measured on diagnosis and approximately every 3 months thereafter using the M-bcr FusionQuant kit (Ipsogen, Marseille, France) according to the manufacturer's protocol. This kit is approved for in vitro diagnostic and it contains the same primers and probes used by the EAC group to quantify the BCR-ABL16Gabert J Beillard E van der Velden VH Bi W Grimwade D Pallisgaard N Barbany G Cazzaniga G Cayuela JM Cavé H Pane F Aerts JL De Micheli D Thirion X Pradel V González M Viehmann S Malec M Saglio G van Dongen JJ Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia—a Europe Against Cancer program.Leukemia. 2003; 17: 2318-2357Crossref PubMed Scopus (1213) Google Scholar and the ABL18Beillard E Pallisgaard N van der Velden VH Bi W Dee R van der Schoot E Delabesse E Macintyre E Gottardi E Saglio G Watzinger F Lion T van Dongen JJ Hokland P Gabert J Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using ‘real-time’ quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR)—a Europe against cancer program.Leukemia. 2003; 17: 2474-2486Crossref PubMed Scopus (718) Google Scholar transcripts, plus a series of controls and calibrators. The performances of the RT-PCR assay were tested by the EAC network,16Gabert J Beillard E van der Velden VH Bi W Grimwade D Pallisgaard N Barbany G Cazzaniga G Cayuela JM Cavé H Pane F Aerts JL De Micheli D Thirion X Pradel V González M Viehmann S Malec M Saglio G van Dongen JJ Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia—a Europe Against Cancer program.Leukemia. 2003; 17: 2318-2357Crossref PubMed Scopus (1213) Google Scholar,18Beillard E Pallisgaard N van der Velden VH Bi W Dee R van der Schoot E Delabesse E Macintyre E Gottardi E Saglio G Watzinger F Lion T van Dongen JJ Hokland P Gabert J Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using ‘real-time’ quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR)—a Europe against cancer program.Leukemia. 2003; 17: 2474-2486Crossref PubMed Scopus (718) Google Scholar briefly the sensitivity was at least of 10−4 dilutions of BCR-ABL-positive RNA in a negative RNA sample, the regression curve had an average slope of −3.45 with R2 >0.95, and the precision of each measurement (ie, the error) was <6% of the corresponding average Ct value. Results of mRNA analysis were reported as the ratio between the number of BCR-ABL and ABL transcripts, with this ratio expressed as a percentage (BCR-ABL/ABL). The sample preparation and the reverse transcription procedures were performed in compliance with the updated international recommendations.17Hughes T Deininger M Hochhaus A Branford S Radich J Kaeda J Baccarani M Cortes J Cross NC Druker BJ Gabert J Grimwade D Hehlmann R Kamel-Reid S Lipton JH Longtine J Martinelli G Saglio G Soverini S Stock W Goldman JM Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for “harmonizing” current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results.Blood. 2006; 108: 28-37Crossref PubMed Scopus (994) Google Scholar Total RNA was extracted with the RNA blood kit (Qiagen, Hilden, Germany) from blood and bone marrow samples previously treated with the HetaSept gradient (Stem Cell Technologies, Vancouver, Canada) to eliminate red cells and erythroid precursors. As part of good laboratory practice, the integrity of RNA was tested by electrophoresis after each extraction; cDNA was synthesized from 1 μg of RNA through the high-efficiency Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA) following the EAC reverse transcription procedure,16Gabert J Beillard E van der Velden VH Bi W Grimwade D Pallisgaard N Barbany G Cazzaniga G Cayuela JM Cavé H Pane F Aerts JL De Micheli D Thirion X Pradel V González M Viehmann S Malec M Saglio G van Dongen JJ Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia—a Europe Against Cancer program.Leukemia. 2003; 17: 2318-2357Crossref PubMed Scopus (1213) Google Scholar as recommended by the M-bcr FusionQuant kit user guide. For each patient, a genomic assay was developed on the basis of his/her BCR-ABL sequence. Each assay comprised two real-time PCR reactions: one directed against the breakpoint sequence (present in one copy, only in leukemic cells) and a second against the BCR sequence used as control (one copy in leukemic cells or two copies in normal cells) (see Supplemental Figure S1 at http://jmd.amjpathol.org). The percentage of leukemic cells (LCs) was calculated using the following formula: LC = 100 · (2/(2ΔCt + 1)), where ΔCt is the difference between the amplification cycles of the BCR-ABL and BCR reactions (see Supplemental Figure S2 at http://jmd.amjpathol.org). Common forward primers and probes (Table 1) were used to ensure that the efficiencies of the two reactions within each assay could be as similar as possible. Plasmids containing the BCR-ABL breakpoint and the correspondent BCR sequence were used as reference material to test the efficiency and sensitivity of each assay. According to the human C value, plasmids were diluted to simulate different concentrations of leukemic DNA starting from 400 ng. For a given assay, the amplification efficiency was considered acceptable when it was higher than 90% in the range from approximately 400 to 0.3 ng for both the BCR-ABL and BCR reactions. The acceptance criteria for the sensitivity was fixed at least 10−4 dilutions (ie, similar to the sensitivity of conventional RQ-PCR assays).16Gabert J Beillard E van der Velden VH Bi W Grimwade D Pallisgaard N Barbany G Cazzaniga G Cayuela JM Cavé H Pane F Aerts JL De Micheli D Thirion X Pradel V González M Viehmann S Malec M Saglio G van Dongen JJ Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia—a Europe Against Cancer program.Leukemia. 2003; 17: 2318-2357Crossref PubMed Scopus (1213) Google Scholar Furthermore, genomic assays were tested for the absence of spurious amplifications after 45 cycles using BCR-ABL negative DNA from healthy subjects.Table 1Primer and Probe SequencesPatient ID1F 5′-CTGCTGCTGGGTGGTTGA-3′Ph-R 5′-GGATTTTAGTCCTTACTTGTTTTCTATTTCAC-3′wt-R 5′-GCCAGATCCAAGGCACAGA-′Probe 6-FAM-AGATGCACGGCTTC-MGB2F 5′-CCCCCTTCCTGTTAGCACTTT-3′Ph-R 5′-GCTGCAACAGTACAAACAGTAACCC-3′wt-R 5′-CCCTAACAAGCATAGCTCTTCCTT-3′Probe 6-FAM-ATGGGACTAGTGGACTTT-MGB3F 5′-GCCCTCCTCTCCTCCAGCTA-3′Ph-R 5′-AAGCCTCTGGCGTGTTTCC-3′wt-R 5′-TGAGCATATGTGCAACAGTGAATG-3′Probe 6-FAM-CACTTTTGGTCAAGCTG-MGB4F 5′-TGGGACTAGTGGACTTTGGTTCA-3′Ph-R 5′-GTGCATGATCATCACTAGTTAAAATGTAAA-3′wt-R 5′-CTAACCCACCTTGTCCACTCCT-3′Probe 6-FAM-ACAAGAGGCCCTAACAA-MGB6F 5′-CACAGCATACGCTATGCACATGT-3′Ph-R 5′-GGGAAAAAATGTTTTCTCCTTATATCG-3′wt-R 5′-ATAAGGTTCCAAGGACAGCAGAG-3′Probe 6-FAM-ACACACACCCCACCC-MGB8F 5′-TGCTCTGTGCCTTGGATCTG-3′Ph-R 5′-TTCGGTGTAAAATCCTTCCATACTTT-3′wt-R 5′-TGCAAAACAGCTTGACCAAAA-3′Probe 6-FAM-CCCCACTCCCGTCCT-MGB9F 5′-TTGTCACCTGCCTCCCTTTC-3′Ph-R 5′-TGAACTCCTGACCTCAAGTGATCT-3′wt-R 5′-GAGCCCCGGAGACTCATCA-3′Probe 6-FAM-CGGGACAACAGAAGC-MGB10F 5′-CACTGGTTTGCCTGTATTGTGAA-3′Ph-R 5′-GGACACACAGGGAACTACACTGC-3′wt-R 5′-TGGGCCAAAAACATACTCATCA-3′Probe 6-FAM-TCCTGAGATCCCC-MGBPR1F 5′-CCGCTGACCATCAATAAGGAA-3′Ph-R 5′-TGCCACGCCTTCTCTTCTG-3′wt-R 5′-CAAAGTCCACTAGTCCCATCAAAA-3′Probe 6-FAM-TTTCCGTGTACAGGGCA-MGBPR2F 5′-TTTTGGTCAAGCTGTTTTGCA-3′Ph-R 5′-GGCACCAGAAGCTGAGTGAAG-3′wt-R 5′-ACACATGTGCATAGCGTATGCTG-3′Probe 6-FAM-TGTTGCACATATGCTC-MGBFor each patient-specific assay, primers are designated as follow: F, common forward primer; Ph-R, reverse primer for the selective amplification of the BCR-ABL sequence; wt-R, reverse primer for the selective amplification of BCR. Open table in a new tab For each patient-specific assay, primers are designated as follow: F, common forward primer; Ph-R, reverse primer for the selective amplification of the BCR-ABL sequence; wt-R, reverse primer for the selective amplification of BCR. Genomic DNA was extracted with the DNA blood Kit (Qiagen) from the same samples used for molecular monitoring of mRNA. The reaction mixture of each real time PCR reaction contained 12.5 μl of TaqMan Universal PCR MasterMix (Applied Biosystems, Foster City, CA); 900 nmol/L each primer; 200 nmol/L probe; DNA ranging from 100 to 300 ng depending on the sample availability; and nuclease-free water up to 25 μl. The PCR thermal profile was 2 minutes at 50°C followed by 10 minutes at 95°C and 45 amplification cycles (95°C for 15 seconds and 60°C for 60 seconds). Reactions were prepared and run in triplicate on ABI Prism 7000 SDS (Applied Biosystems) and each experiment was repeated and confirmed a second time. The presence of Ph chromosomes was evident in 100% of the bone marrow metaphases of each patient at diagnosis. The majority of patients (8 of 10) had a complete response within 6 months from the beginning of the treatment, and the results of subsequent cytogenetic investigations were negative. Only patients 6 and 10 still had evidence of the BCR-ABL translocation at 12 and 6 months, respectively; a stable cytogenetic remission was achieved and maintained after the dose of IM was increased to 600 mg/day. For each patient, a genomic assay was developed fulfilling the requirements for efficiency, sensitivity, and absence of spurious amplifications as established in Materials and Methods (see Supplemental Figures S3-S4 at http://jmd.amjpathol.org). At the onset, the median baseline level of BCR-ABL mRNA reported as BCR-ABL/ABL was 164% (range, 60% to 280%, SD 77), whereas the average percentage of BCR-ABL positive cells quantified with the genomic approach was 81% (range, 35% to 95%, SD 19). Only patient 1 had a proportion of leukemic cells at the diagnosis (35%) that was significantly different from the average value of 81% (see Supplemental Figure S5 at http://jmd.amjpathol.org). For the time points with both results available, the actual percentage of leukemic cells and the corresponding levels of BCR ABL mRNA measured during follow-up are reported in Table 2. For each of the 57 samples, the extent of sample degradation was evaluated by electrophoresis and PCR amplifications. The average Ct values of the reference sequences (ABL for the RNA samples and BCR for the DNA samples) were 24.63 (range, 22.85 to 25.82) and 22.42 (range, 21.71 to 23.54), respectively. These findings are consistent with the results of electrophoresis and confirm the integrity of the nucleic acids analyzed in this study.Table 2Levels of Residual Disease (%) Measured by Simultaneous mRNA and DNA AnalysisMonths of therapy from the diagnosisPatient no.0361215182124273033421228*Bone marrow samples.2.730†Peripheral blood samples.0.050*Bone marrow samples.UND†Peripheral blood samples.——UND*Bone marrow samples.—————35*Bone marrow samples.0.077†Peripheral blood samples.0.004*Bone marrow samples.UND†Peripheral blood samples.——UND*Bone marrow samples.—————2264*Bone marrow samples.1.630†Peripheral blood samples.0.180†Peripheral blood samples.————UND*Bone marrow samples.————87*Bone marrow samples.1.064†Peripheral blood samples.0.391†Peripheral blood samples.————UND*Bone marrow samples.————3144*Bone marrow samples.—0.920*Bone marrow samples.—0.200†Peripheral blood samples.0.180*Bone marrow samples.0.060*Bone marrow samples.—————94*Bone ma" @default.
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• W2005706794 date "2009-09-01" @default.
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• W2005706794 title "Molecular Monitoring of Residual Disease in Chronic Myeloid Leukemia by Genomic DNA Compared with Conventional mRNA Analysis" @default.
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