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• W2593649206 abstract "Nested RT-PCR (nPCR) and real-time quantitative PCR (qPCR) are well-established methods for monitoring minimal residual disease (MRD) in acute promyelocytic leukemia (APL). Despite their remarkable sensitivity and specificity, both methods have inherent limitations, such as qualitative MRD evaluation and relative quantification. Herein, we used droplet digital PCR (ddPCR) to monitor MRD in 21 APL patients and compared its performance with nPCR and qPCR. After assessing the limit of detection (LOD) for each technique on serial dilutions of PML-RARA bcr1 and bcr3 transcripts, a total of 48 follow-up samples were analyzed and the results compared. ddPCR showed good linearity and efficiency and reached an LOD comparable or even superior to nPCR and qPCR. When tested on primary samples, ddPCR exhibited a sensitivity and specificity of ≥95% and ≥91% for bcr1 and bcr3 transcripts and displayed a significant concordance with both techniques, particularly with nPCR. The peculiar advantage of ddPCR-based monitoring of MRD is represented by absolute quantification, which provides crucial information for the management of patients whose MRD fluctuates under the LOD of qPCR and is detectable, but not quantifiable, by nPCR. Our findings highlight ddPCR as a reliable complementary approach to monitor MRD in APL, and suggest its advantageous application, particularly for the molecular follow-up of patients at high risk of relapse. Nested RT-PCR (nPCR) and real-time quantitative PCR (qPCR) are well-established methods for monitoring minimal residual disease (MRD) in acute promyelocytic leukemia (APL). Despite their remarkable sensitivity and specificity, both methods have inherent limitations, such as qualitative MRD evaluation and relative quantification. Herein, we used droplet digital PCR (ddPCR) to monitor MRD in 21 APL patients and compared its performance with nPCR and qPCR. After assessing the limit of detection (LOD) for each technique on serial dilutions of PML-RARA bcr1 and bcr3 transcripts, a total of 48 follow-up samples were analyzed and the results compared. ddPCR showed good linearity and efficiency and reached an LOD comparable or even superior to nPCR and qPCR. When tested on primary samples, ddPCR exhibited a sensitivity and specificity of ≥95% and ≥91% for bcr1 and bcr3 transcripts and displayed a significant concordance with both techniques, particularly with nPCR. The peculiar advantage of ddPCR-based monitoring of MRD is represented by absolute quantification, which provides crucial information for the management of patients whose MRD fluctuates under the LOD of qPCR and is detectable, but not quantifiable, by nPCR. Our findings highlight ddPCR as a reliable complementary approach to monitor MRD in APL, and suggest its advantageous application, particularly for the molecular follow-up of patients at high risk of relapse. CME Accreditation Statement: This activity (“The JMD 2017 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“The JMD 2017 CME Program in Molecular Diagnostics”) for a maximum of 36 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity (“The JMD 2017 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“The JMD 2017 CME Program in Molecular Diagnostics”) for a maximum of 36 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Acute promyelocytic leukemia (APL) is a rare hematological malignancy commonly associated with the chromosomal translocation t(15;17)(q24;q21), which involves the promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARA) genes, resulting in the oncogenic fusion transcript PML-RARA.1Lo-Coco F. Hasan S.K. Understanding the molecular pathogenesis of acute promyelocytic leukemia.Best Pract Res Clin Haematol. 2014; 27: 3-9Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Although the breakpoints on chromosome 17 are localized within a 17-kb fragment of the RARA intron 2, up to three regions of the PML locus may be involved in the translocation: intron 6, exon 6, and intron 3, accounting for 55%, 5%, and 40% of cases, respectively. The different breakpoints lead to three possible PML-RARA isoforms, referred to as long (L or bcr1), variant (V or bcr2), and short (S or bcr3).2Biondi A. Rambaldi A. Pandolfi P.P. Rossi V. Giudici G. Alcalay M. Lo Coco F. Diverio D. Pogliani E.M. Lanzi E.M. Molecular monitoring of the myl/retinoic acid receptor-alpha fusion gene in acute promyelocytic leukemia by polymerase chain reaction.Blood. 1992; 80: 492-497PubMed Google Scholar Notably, the bcr3 isoform is associated with two well-established adverse prognostic factors (ie, higher white cell counts and the M3 variant morphology).3Albano F. Mestice A. Pannunzio A. Lanza F. Martino B. Pastore D. Ferrara F. Carluccio P. Nobile F. Castoldi G. Liso V. Specchia G. The biological characteristics of CD34+ CD2+ adult acute promyelocytic leukemia and the CD34 CD2 hypergranular (M3) and microgranular (M3v) phenotypes.Haematologica. 2006; 91: 311-316PubMed Google Scholar Current treatment is highly successful, leading to long-term remission and possibly the cure for approximately 70% of newly diagnosed patients.4Tallman M.S. Andersen J.W. Schiffer C.A. Appelbaum F.R. Feusner J.H. Woods W.G. Ogden A. Weinstein H. Shepherd L. Willman C. Bloomfield C.D. Rowe J.M. Wiernik P.H. All-trans retinoic acid in acute promyelocytic leukemia: long-term outcome and prognostic factor analysis from the North American Intergroup protocol.Blood. 2002; 100: 4298-4302Crossref PubMed Scopus (381) Google Scholar, 5Sanz M.A. Martin G. Rayon C. Esteve J. Gonzalez M. Diaz-Mediavilla J. Bolufer P. Barragan E. Terol M.J. Gonzalez J.D. Colomer D. Chillon C. Rivas C. Gomez T. Ribera J.M. Bornstein R. Roman J. Calasanz M.J. Arias J. Alvarez C. Ramos F. Deben G. PETHEMA GroupA modified AIDA protocol with anthracycline-based consolidation results in high antileukemic efficacy and reduced toxicity in newly diagnosed PML/RARalpha-positive acute promyelocytic leukemia.Blood. 1999; 94: 3015-3021PubMed Google Scholar, 6Lo Coco F. Diverio D. Falini B. Biondi A. Nervi C. Pelicci P.G. Genetic diagnosis and molecular monitoring in the management of acute promyelocytic leukemia.Blood. 1999; 94: 12-22Crossref PubMed Google Scholar, 7Burnett A.K. Grimwade D. Solomon E. Wheatley K. Goldstone A.H. Presenting white blood cell count and kinetics of molecular remission predict prognosis in acute promyelocytic leukemia treated with all-trans retinoic acid: result of the Randomized MRC Trial.Blood. 1999; 93: 4131-4143Crossref PubMed Google Scholar, 8Fenaux P. Chastang C. Chevret S. Sanz M. Dombret H. Archimbaud E. Fey M. Rayon C. Huguet F. Sotto J.J. Gardin C. Makhoul P.C. Travade P. Solary E. Fegueux N. Bordessoule D. Miguel J.S. Link H. Desablens B. Stamatoullas A. Deconinck E. Maloisel F. Castaigne S. Preudhomme C. Degos L. European APL GroupA randomized comparison of all transretinoic acid (ATRA) followed by chemotherapy and ATRA plus chemotherapy and the role of maintenance therapy in newly diagnosed acute promyelocytic leukemia.Blood. 1999; 94: 1192-1200PubMed Google Scholar However, a small group of patients are at particular risk of relapse, which is not predictable on the basis of clinical parameters, and may potentially benefit from an early assessment of the minimal residual disease (MRD).2Biondi A. Rambaldi A. Pandolfi P.P. Rossi V. Giudici G. Alcalay M. Lo Coco F. Diverio D. Pogliani E.M. Lanzi E.M. Molecular monitoring of the myl/retinoic acid receptor-alpha fusion gene in acute promyelocytic leukemia by polymerase chain reaction.Blood. 1992; 80: 492-497PubMed Google Scholar, 3Albano F. Mestice A. Pannunzio A. Lanza F. Martino B. Pastore D. Ferrara F. Carluccio P. Nobile F. Castoldi G. Liso V. Specchia G. The biological characteristics of CD34+ CD2+ adult acute promyelocytic leukemia and the CD34 CD2 hypergranular (M3) and microgranular (M3v) phenotypes.Haematologica. 2006; 91: 311-316PubMed Google Scholar, 9Sanz M.A. Lo Coco F. Martin G. Avvisati G. Rayon C. Barbui T. Diaz-Mediavilla J. Fioritoni G. Gonzalez J.D. Liso V. Esteve J. Ferrara F. Bolufer P. Bernasconi C. Gonzalez M. Rodeghiero F. Colomer D. Petti M.C. Ribera J.M. Mandelli F. Definition of relapse risk and role of nonanthracycline drugs for consolidation in patients with acute promyelocytic leukemia: a joint study of the PETHEMA and GIMEMA cooperative groups.Blood. 2000; 96: 1247-1253PubMed Google Scholar, 10Santamaria C. Chillon M.C. Fernandez C. Martin-Jimenez P. Balanzategui A. Garcia Sanz R. San Miguel J.F. Gonzalez M.G. Using quantification of the PML-RARalpha transcript to stratify the risk of relapse in patients with acute promyelocytic leukemia.Haematologica. 2007; 92: 315-322Crossref PubMed Scopus (61) Google Scholar, 11Breccia M. Mandelli F. Petti M.C. D'Andrea M. Pescarmona E. Pileri S.A. Carmosino I. Russo E. De Fabritiis P. Alimena G. Clinico-pathological characteristics of myeloid sarcoma at diagnosis and during follow-up: report of 12 cases from a single institution.Leuk Res. 2004; 28: 1165-1169Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Tobal K. Moore H. Macheta M. Yin J.A. Monitoring minimal residual disease and predicting relapse in APL by quantitating PML-RARalpha transcripts with a sensitive competitive RT-PCR method.Leukemia. 2001; 15: 1060-1065Crossref PubMed Scopus (24) Google Scholar, 13Jurcic J.G. Nimer S.D. Scheinberg D.A. DeBlasio T. Warrell Jr., R.P. Miller Jr., W.H. Prognostic significance of minimal residual disease detection and PML/RAR-alpha isoform type: long-term follow-up in acute promyelocytic leukemia.Blood. 2001; 98: 2651-2656Crossref PubMed Scopus (86) Google Scholar For this reason, the detection of the PML-RARA transcript, performed at the post-consolidation phase, provides an independent prognostic factor in APL.14Grimwade D. Jovanovic J.V. Hills R.K. Nugent E.A. Patel Y. Flora R. Diverio D. Jones K. Aslett H. Batson E. Rennie K. Angell R. Clark R.E. Solomon E. Lo-Coco F. Wheatley K. Burnett A.K. Prospective minimal residual disease monitoring to predict relapse of acute promyelocytic leukemia and to direct pre-emptive arsenic trioxide therapy.J Clin Oncol. 2009; 27: 3650-3658Crossref PubMed Scopus (260) Google Scholar, 15Gallagher R.E. Yeap B.Y. Bi W. Livak K.J. Beaubier N. Rao S. Bloomfield C.D. Appelbaum F.R. Tallman M.S. Slack J.L. Willman C.L. Quantitative real-time RT-PCR analysis of PML-RAR alpha mRNA in acute promyelocytic leukemia: assessment of prognostic significance in adult patients from intergroup protocol 0129.Blood. 2003; 101: 2521-2528Crossref PubMed Scopus (128) Google Scholar PML-RARA amplification by qualitative RT-PCR is the method most commonly used to confirm the morphological diagnosis of APL and is essential for defining the PML breakpoint location and establishing the target for reliable molecular monitoring.16van 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. Diaz 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 (1006) Google Scholar Nested RT-PCR (nPCR) was widely used for MRD evaluation, despite the disadvantage of providing unreliable results (ie, MRD positivity even in long-term remission patients who never experience a further hematological relapse).17Tobal K. Liu Yin J.A. RT-PCR method with increased sensitivity shows persistence of PML-RARA fusion transcripts in patients in long-term remission of APL.Leukemia. 1998; 12: 1349-1354Crossref PubMed Scopus (42) Google Scholar This limitation, together with the need for a precise quantification of the transcript, led to the introduction of real-time quantitative PCR (qPCR), which is now the method generally used to monitor MRD in APL.18Gabert J. Beillard E. van der Velden V.H. Bi W. Grimwade D. Pallisgaard N. Barbany G. Cazzaniga G. Cayuela J.M. Cave H. Pane F. Aerts J.L. De Micheli D. Thirion X. Pradel V. Gonzalez M. Viehmann S. Malec M. Saglio G. van Dongen J.J. 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 (1228) Google Scholar qPCR offers several advantages compared to nPCR, such as a higher sensitivity, reduced risk of contamination,19Flora R. Grimwade D. Real-time quantitative RT-PCR to detect fusion gene transcripts associated with AML.Methods Mol Med. 2004; 91: 151-173PubMed Google Scholar and the possibility of monitoring the quality of samples (by the amplification of a housekeeping gene) and following the disease kinetics.6Lo Coco F. Diverio D. Falini B. Biondi A. Nervi C. Pelicci P.G. Genetic diagnosis and molecular monitoring in the management of acute promyelocytic leukemia.Blood. 1999; 94: 12-22Crossref PubMed Google Scholar, 20Grimwade D. Lo Coco F. Acute promyelocytic leukemia: a model for the role of molecular diagnosis and residual disease monitoring in directing treatment approach in acute myeloid leukemia.Leukemia. 2002; 16: 1959-1973Crossref PubMed Scopus (159) Google Scholar The major limitation of qPCR is represented by relative quantification, and most important, an inadequate quantification of samples that have a tumor burden between the sensitivity threshold and the quantitative range of the technique.21van der Velden V.H. Panzer-Grumayer E.R. Cazzaniga G. Flohr T. Sutton R. Schrauder A. Basso G. Schrappe M. Wijkhuijs J.M. Konrad M. Bartram C.R. Masera G. Biondi A. van Dongen J.J. Optimization of PCR-based minimal residual disease diagnostics for childhood acute lymphoblastic leukemia in a multi-center setting.Leukemia. 2007; 21: 706-713Crossref PubMed Scopus (123) Google Scholar Nanoliter-sized droplet technology paired with droplet digital PCR (ddPCR) is a direct method for the precise and absolute quantification of nucleic acids, based on limiting partition of the PCR volume and on Poisson statistics.22Pinheiro L.B. Coleman V.A. Hindson C.M. Herrmann J. Hindson B.J. Bhat S. Emslie K.R. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification.Anal Chem. 2012; 84: 1003-1011Crossref PubMed Scopus (710) Google Scholar, 23Sykes P.J. Neoh S.H. Brisco M.J. Hughes E. Condon J. Morley A.A. Quantitation of targets for PCR by use of limiting dilution.Biotechniques. 1992; 13: 444-449PubMed Google Scholar Being independent of a reference standard curve and allowing for high-sensitive absolute quantification of the target, ddPCR could have a high potential in monitoring MRD. Herein, we investigate whether ddPCR could overcome some of the above-mentioned limitations of nPCR and qPCR. We, therefore, compared the performances of these three techniques both on reference dilutions and primary samples, to seek for the most appropriate and reliable technology for the molecular monitoring of MRD in APL. The APL patients included in this report were treated according to the AIDA2000 GIMEMA group protocol.24Lo-Coco F. Avvisati G. Vignetti M. Breccia M. Gallo E. Rambaldi A. Paoloni F. Fioritoni G. Ferrara F. Specchia G. Cimino G. Diverio D. Borlenghi E. Martinelli G. Di Raimondo F. Di Bona E. Fazi P. Peta A. Bosi A. Carella A.M. Fabbiano F. Pogliani E.M. Petti M.C. Amadori S. Mandelli F. Italian G.C.G. Front-line treatment of acute promyelocytic leukemia with AIDA induction followed by risk-adapted consolidation for adults younger than 61 years: results of the AIDA-2000 trial of the GIMEMA Group.Blood. 2010; 116: 3171-3179Crossref PubMed Scopus (235) Google Scholar The diagnosis was initially morphological and was confirmed by detection of the PML-RARA fusion gene, as reported.16van 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. Diaz 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 (1006) Google Scholar ddPCR was compared head-to-head with nPCR and qPCR in 21 patients, 11 bearing the bcr1 transcript and 10 bearing the bcr3 transcript, for a total of 48 follow-up samples, including replicates. Patients with at least 12 months of follow-up from the end of consolidation therapy were selected, and grouped on the basis of their relapse risk score (Sanz score) and disease course. The bcr1-positive patients were assigned either to the complete hematological remission (CHR; n = 5) or to the molecular or hematological relapse/relapse-risk group (R/RR; n = 6). Similarly, four and six bcr3-positive patients were assigned to the CHR and the relapse (R) group, respectively. The study protocol was approved by the local ethics committee, and all patients provided written informed consent to take part in the study. Technique performances were evaluated on serial dilutions of cell lines and patients' bone marrow RNA. NB4 and HL60 cell lines (DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 1% l-glutamine, and 1% antibiotics (penicillin/streptomycin; 100 U/mL) (all from EuroClone S.p.A., Milan, Italy) in a 5% CO2-enriched atmosphere at 37°C. RNA was extracted with the Qiacube automated extraction system using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and quantified by a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA). To generate proper standard curves, 1 μg of RNA from NB4 cells, which have the PML breakpoint in the bcr1 region, or 1 μg of RNA from a primary sample, with the PML breakpoint in the bcr3 region, was serially 10-fold diluted in RNA from HL-60 cells (negative for both the transcripts). Each sample was reverse-transcribed using the QuantiTect reverse transcription kit (Qiagen) in triplicates, and triplicates were pooled before molecular analyses. To minimize possible biases related to sampling, all PCR experiments were performed on the same cDNA, after pooling the reverse-transcribed triplicates. nPCR was performed according to the BIOMED-1 concerted action report.16van 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. Diaz 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 (1006) Google Scholar Only samples showing amplification of the housekeeping gene β-actin (ACTB) were further investigated for the presence of the transcript of interest. The first round of nPCR was performed on 100 ng of cDNA, whereas the second round was conducted on 1 μL of the first round reaction, in triplicates. Samples displaying nPCR positivity were further analyzed by Sanger sequencing of the amplification product, to confirm the presence of the rearrangement. qPCR was performed on a LightCycler II 480 system (Roche Diagnostics, Monza, Italy), with the Ipsogen PML-RARA bcr1 and bcr3 IVD kits (Qiagen). MRD estimation was based on five plasmid 10-fold standard dilutions for the bcr1 and bcr3 transcripts, and on three plasmid standard dilutions for the ABL proto-oncogene 1 (ABL1) control gene. MRD analysis was conducted starting from 100 ng of cDNA in duplicates, and results were interpreted according to the manufacturer's instructions. In particular, samples with an ABL1 copy number <1318 were classified as not analyzable and excluded from further analyses. ddPCR for both PML-RARA bcr1 and bcr3 isoforms was performed with primers and probes, as previously described.25Albano F. Zagaria A. Anelli L. Coccaro N. Tota G. Brunetti C. Minervini C.F. Impera L. Minervini A. Cellamare A. Orsini P. Cumbo C. Casieri P. Specchia G. Absolute quantification of the pretreatment PML-RARA transcript defines the relapse risk in acute promyelocytic leukemia.Oncotarget. 2015; 6: 13269-13277Crossref PubMed Scopus (32) Google Scholar Briefly, glucuronidase β (GUSB) was used to assess the quality of cDNA samples; in addition, because of the need to test a large amount of material, PML-RARA and GUSB transcripts were tested in separate reactions. PML-RARA primers and probes were used at the final concentrations of 900 and 250 nmol/L, respectively, and 200 ng of cDNA template was used in a final volume of 20 μL. Eight wells were analyzed per patient. Droplets were obtained with a QX200 droplet generator, and amplification was performed on a T100 Thermal Cycler (all from Bio-Rad, Hercules, CA). PCR products were run on a QX200 droplet reader (Bio-Rad), analyzed with QuantaSoft software version 1.7.4 (Bio-Rad), and the quantification of the target concentration was presented as PML-RARA copies per microliter. In ddPCR, droplets are assigned as positive or negative by thresholding based on their fluorescence amplitude. The bcr1 and bcr3 probes displayed different fluorescence amplitudes; therefore, thresholds were set differently for the two assays (3100 for bcr1 and 4100 for bcr3). Each experiment included a negative (healthy donor) and a positive control sample (previously tested diagnostic specimen), as well as a no-template control. Results were analyzed when the number of accepted droplets per well was at least 10,000 and the housekeeping gene provided correct (expected number of copies/μL) and reproducible amplification. Bcr1 and bcr3 ddPCR linear regression curves were calculated from the standard dilution, whereas the amplification efficiency was calculated according to the following equation: E=10(−1/slope)−1. Detailed information is provided in Supplemental Table S1, as suggested by the guidelines for the Minimum Information for the Publication of Digital PCR Experiments.26Huggett J.F. Foy C.A. Benes V. Emslie K. Garson J.A. Haynes R. Hellemans J. Kubista M. Mueller R.D. Nolan T. Pfaffl M.W. Shipley G.L. Vandesompele J. Wittwer C.T. Bustin S.A. The digital MIQE guidelines: minimum Information for Publication of Quantitative Digital PCR Experiments.Clin Chem. 2013; 59: 892-902Crossref PubMed Scopus (594) Google Scholar The limit of detection (LOD) for ddPCR was measured according to the Clinical and Laboratory Standards Institute guidelines EP17.27Tholen D.W. Linnet K. Kondratovich M. Armbruster D.A. Garrett P.E. Jones R.L. Kroll M.H. Lequin R.M. Pankratz T.J. Scassellati G.A. Schimmel H. Tsai J. Clinical and Laboratory Standard InstituteProtocols for Determination of limits of Detection and Limits of Quantitation. Approved guideline EP17-A. Clinical and Laboratory Standard Institute, Wayne, PA2004Google Scholar Briefly, after defining the limit of blank (LOB), multiple replicates of samples containing increasingly lower concentrations of the analyte were assessed according to the following formula: LOD=LOB+Cβ×σS, where LOB corresponds to the 95th percentile of the distribution of the blank values: Cβ=1645/[1−1(4×f)], f = degree of freedom of the estimated σS calculated as: f=Ns−K (Ns, number of samples; K, number of measurements), and σS is the SD of the sample with the lowest concentration. For the LOB, 20 replicates of 100% HL-60 cDNA were tested, whereas for the LOD the number of replicates was dependent on the specific dilution analyzed, ranging from a minimum of two (as for 100% NB4 cDNA) up to eight replicates (0.001% NB4 cDNA). For both ddPCR and qPCR, the LOD was accepted when three experimental replicates provided positive results in 100% of cases. Correlation analysis between ddPCR and qPCR was calculated by the Pearson test. Sensitivity was calculated according to the following formula: TP/(TP+FN), and specificity was calculated as follows: TN/(TN+FP), TP indicates true positive; FN, false negative; TN, true negative; and FP, false positive. CIs were calculated by the Wilson/Brown method. For the comparison of quantitative variables, the Mann-Whitney test or the Kruskal-Wallis test was used. All tests were two-tailed, and statistical significance was set at P ≤ 0.05. Statistical parameters were calculated using GraphPad Prism software version 7.0 (GraphPad Software Inc., La Jolla, CA). The LOB was calculated as 95th percentile of the distribution of the false-positive values and resulted in 0.007 copies/μL. The LOD, calculated on sequential NB4 in HL-60 RNA dilutions, was 0.01% for both the bcr1 (Figure 1, A and C) and bcr3 (Figure 1, B and D) transcripts, and more precisely resulted in 0.15 and 0.13 copies/μL for bcr1 and bcr3, respectively. The linearity of ddPCR was assessed by quantifying 10-fold–diluted PML-RARA bcr1 or bcr3 transcripts (standard dilutions). In both cases, the curves showed a good linearity (R2 = 0.99) and efficiency (E) (ie, E = 92% for bcr1 and E = 91% for bcr3) (Figure 2, A and B).Figure 2PML-RARA quantification by nPCR, qPCR, and ddPCR. Graphics show log10-transformed standard dilutions plotted against the corresponding log10-transformed cDNA copy numbers of bcr1 (A) and bcr3 (B) transcripts obtained by ddPCR and fitted with a linear regression model. C: Point-to-point comparison of ddPCR and qPCR results. The log10-transformed standard dilutions were plotted against the corresponding log10-transformed cDNA copy numbers determined by ddPCR (open gray dots) and qPCR (closed black dots). The dotted line indicates the qPCR quantification limit. E, efficiency.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The same standard dilutions were also tested by nPCR and qPCR. The LOD for nPCR was 0.01% (Table 1), whereas in our experimental conditions qPCR allowed a detection as low as 0.1% of both transcripts (Table 1 and Figure 2C). Comparison of qPCR and ddPCR showed a good correlation for high copy number samples (Pearson r = 0.99, P < 0.001), whereas for lower concentrations this correlation was not assessable because of the detection limit of qPCR (0.1%). Overall, nPCR and ddPCR showed the highest rate of concordance for each standard dilution assessed, for both transcripts (Table 1).Table 1LODs of nPCR, qPCR, and ddPCRPML-RARA bcr1 and bcr3100%10%1%0.1%0.01%0.001%0%nPCR+++++−−qPCR++++±∗Positive results were obtained in 50% of evaluations.−−ddPCR+++++±∗Positive results were obtained in 50% of evaluations.−+, positive; −, negative; LOD, limit of detection.∗ Positive results were obtained in 50% of evaluations. Open table in a new tab +, positive; −, negative; LOD, limit of detection. Consistent with these data, we identified a bcr1-positive patient for whom the detection of the fusion gene transcript was achieved only by nPCR and ddPCR (Figure 3, A and C), whereas no amplification was obtained by qPCR (Figure 3B). On the basis of the absolute transcript copy number, the MRD load was estimated to be comparable to the 0.01% standard dilution. All samples were assayed by nPCR, whereas qPCR was performed in 96% of cases (46/48); 11% of these (5/46) were not interpretable because the ABL1 copy numbers did not reach the recommended threshold; ddPCR was performed in 100% of cases (48/48). On the basis of the different LODs calculated for nPCR and qPCR, we considered the former as the reference method for interpreting ddPCR results (LOD = 0.01% versus 0.1%). Of 19 follow-up samples bcr1-positive by nPCR, 95% (18/19) were also positive by ddPCR, and 23% (3/13) by qPCR. Of the 11 cases that resulted bcr1-negative by nPCR, 91% (10/11) were also negative by ddPCR, and 100% (10/10) by qPCR. ddPCR sensitivity for the bcr1 transcript was estimated at 0.95 (95% CI, 0.75–0.99), whereas specificity was 0.91 (95% CI, 0.62–0.99) (Figure 4A). Moreover, we found that 100% (6/6) of the bcr3 samples that" @default.
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• W2593649206 title "Droplet Digital PCR Is a Reliable Tool for Monitoring Minimal Residual Disease in Acute Promyelocytic Leukemia" @default.
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