Digital Droplet PCR Is a Reliable Tool to Improve Minimal Residual Disease Stratification in Adult Philadelphia-Negative Acute Lymphoblastic Leukemia

Abstract

Digital droplet PCR (ddPCR) is an implementation of conventional PCR, with the potential of overcoming some limitations of real-time quantitative PCR (RQ-PCR). To evaluate if ddPCR may improve the quantification of disease levels and refine patients' risk stratification, 116 samples at four time points from 44 (35 B-lineage and 9 T-lineage) adult Philadelphia-negative acute lymphoblastic leukemia patients enrolled in the GIMEMA LAL1913 protocol were analyzed by RQ-PCR and ddPCR. A concordance rate between RQ-PCR and ddPCR of 79% (P < 0.0001) was observed; discordances were identified in 21% of samples, with the majority being RQ-PCR-negative (NEG) or positive not quantifiable (PNQ). ddPCR significantly reduced the proportion of PNQ samples—2.6% versus 14% (P = 0.003)—and allowed disease quantifiability in 6.6% of RQ-PCR-NEG, increasing minimal residual disease quantification in 14% of samples. Forty-seven samples were also investigated by next-generation sequencing, which confirmed the ddPCR results in samples classified as RQ-PCR-PNQ or NEG. By reclassifying samples on the basis of the ddPCR results, a better event-free survival stratification of patients was observed compared to RQ-PCR; indeed, ddPCR captured more true-quantifiable samples, with five relapses occurring in three patients who resulted RQ-PCR-PNQ/NEG but proved ddPCR positive quantifiable. At variance, no relapses were recorded in patients whose follow-up samples were RQ-PCR-PNQ but reclassified as ddPCR-NEG. A broader application of ddPCR in acute lymphoblastic leukemia clinical trials will help to improve patients' stratification. Digital droplet PCR (ddPCR) is an implementation of conventional PCR, with the potential of overcoming some limitations of real-time quantitative PCR (RQ-PCR). To evaluate if ddPCR may improve the quantification of disease levels and refine patients' risk stratification, 116 samples at four time points from 44 (35 B-lineage and 9 T-lineage) adult Philadelphia-negative acute lymphoblastic leukemia patients enrolled in the GIMEMA LAL1913 protocol were analyzed by RQ-PCR and ddPCR. A concordance rate between RQ-PCR and ddPCR of 79% (P < 0.0001) was observed; discordances were identified in 21% of samples, with the majority being RQ-PCR-negative (NEG) or positive not quantifiable (PNQ). ddPCR significantly reduced the proportion of PNQ samples—2.6% versus 14% (P = 0.003)—and allowed disease quantifiability in 6.6% of RQ-PCR-NEG, increasing minimal residual disease quantification in 14% of samples. Forty-seven samples were also investigated by next-generation sequencing, which confirmed the ddPCR results in samples classified as RQ-PCR-PNQ or NEG. By reclassifying samples on the basis of the ddPCR results, a better event-free survival stratification of patients was observed compared to RQ-PCR; indeed, ddPCR captured more true-quantifiable samples, with five relapses occurring in three patients who resulted RQ-PCR-PNQ/NEG but proved ddPCR positive quantifiable. At variance, no relapses were recorded in patients whose follow-up samples were RQ-PCR-PNQ but reclassified as ddPCR-NEG. A broader application of ddPCR in acute lymphoblastic leukemia clinical trials will help to improve patients' stratification. Philadelphia-negative acute lymphoblastic leukemia (Ph− ALL) represents ≥70% of all adult ALL cases. Although the use of pediatric-inspired ALL treatment strategies has led to substantial improvements, about 50% of adults still relapse1Fielding A.K. Richards S.M. Chopra R. Lazarus H.M. Litzow M.R. Buck G. Durrant I.J. Luger S.M. Marks D.I. Franklin I.M. McMillan A.K. Tallman M.S. Rowe J.M. Goldstone A.H. Medical Research Council of the United Kingdom Adult ALL Working Party; Eastern Cooperative Oncology GroupOutcome of 609 adults after relapse of acute lymphoblastic leukemia (ALL); an MRC UKALL12/ECOG 2993 study.Blood. 2007; 109: 944-950Crossref PubMed Scopus (581) Google Scholar,2Gökbuget N. Stanze D. Beck J. Diedrich H. Horst H.A. Hüttmann A. Kobbe G. Kreuzer K.A. Leimer L. Reichle A. Schaich M. Schwartz S. Serve H. Starck M. Stelljes M. Stuhlmann R. Viardot A. Wendelin K. Freund M. Hoelzer D. German Multicenter Study Group for Adult Acute Lymphoblastic LeukemiaOutcome of relapsed adult lymphoblastic leukemia depends on response to salvage chemotherapy, prognostic factors, and performance of stem cell transplantation.Blood. 2012; 120: 2032-2041Crossref PubMed Scopus (294) Google Scholar. There is broad evidence that minimal residual disease (MRD) represents a major prognostic indicator; clinical protocols for childhood and adult ALL are tailored according to MRD assessments at different time points (TPs). Currently, real-time quantitative PCR (RQ-PCR) of clonotypic immunoglobulin and T-cell receptor (IG/TR) gene rearrangements is the most widely used molecular method for MRD assessment, standardized according to the EuroMRD consortium guidelines.3van der Velden V.H. Cazzaniga G. Schrauder A. Hancock J. Bader P. Panzer-Grumayer E.R. Flohr T. Sutton R. Cave H. Madsen H.O. Cayuela J.M. Trka J. Eckert C. Foroni L. Zur Stadt U. Beldjord K. Raff T. van der Schoot C.E. van Dongen J.J.M. European Study Group on MRD detection in ALL (ESG-MRD-ALL)Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data.Leukemia. 2007; 21: 604-611Crossref PubMed Scopus (535) Google Scholar However, non-specific amplifications of spurious IG/TR gene rearrangements are hardly distinguishable from positive cases at a low level [positive not quantifiable (PNQ)] by RQ-PCR, with an intrinsic risk of false-positive/negative MRD detections. These cases are thus troublesome to interpret, representing a major challenge in the monitoring of patients at a time when MRD is incorporated in clinical trials and is guiding treatment decisions. Moreover, the use of RQ-PCR can be limited by the lack of sufficient diagnostic material because the method is based on the comparison, for each experiment, with a standard curve based on diagnostic neoplastic DNA, thus limiting the possibility of monitoring patients over time. Digital droplet PCR (ddPCR) and next-generation sequencing (NGS) are advanced molecular methods, investigated within the European Scientific Foundation for Laboratory Hemato-Oncology (ESLHO), in the EuroClonality and EuroMRD Consortium groups, that could help to overcome the limits of RQ-PCR and potentially provide a more precise definition of the MRD status.4Whale A.S. Huggett J.F. Cowen S. Speirs V. Shaw J. Ellison S. Foy C.A. Scott D.J. Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation.Nucleic Acids Res. 2012; 40: e82Crossref PubMed Scopus (312) Google Scholar, 5Hindson C.M. Chevillet J.R. Briggs H.A. Gallichotte E.N. Ruf I.K. Hindson B.J. Vessella R.L. Tewari M. Absolute quantification by droplet digital PCR versus analog real-time PCR.Nat Methods. 2013; 10: 1003-1005Crossref PubMed Scopus (876) Google Scholar, 6Kotrova M. van der Velden V.H. van Dongen J.J. Formankova R. Sedlacek P. Brüggemann M. Zuna J. Stary J. Trka J. Fronkova E. Next-generation sequencing indicates false-positive MRD results and better predicts prognosis after SCT in patients with childhood ALL.Bone Marrow Transplant. 2017; 52: 962-968Crossref PubMed Scopus (57) Google Scholar The ddPCR technology is an implementation of conventional PCR that allows the quantitation of nucleic acid targets without the need of the calibration curves.7Sanders R. Huggett J.F. Bushell C.A. Cowen S. Scott D.J. Foy C.A. Evaluation of digital PCR for absolute DNA quantification.Anal Chem. 2011; 83: 6474-6484Crossref PubMed Scopus (247) Google Scholar It has a sensitivity, accuracy, and reproducibility at least comparable to that of RQ-PCR and shows a good analytical performance to quantify low positive samples defined as PNQ by RQ-PCR,4Whale A.S. Huggett J.F. Cowen S. Speirs V. Shaw J. Ellison S. Foy C.A. Scott D.J. Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation.Nucleic Acids Res. 2012; 40: e82Crossref PubMed Scopus (312) Google Scholar,5Hindson C.M. Chevillet J.R. Briggs H.A. Gallichotte E.N. Ruf I.K. Hindson B.J. Vessella R.L. Tewari M. Absolute quantification by droplet digital PCR versus analog real-time PCR.Nat Methods. 2013; 10: 1003-1005Crossref PubMed Scopus (876) Google Scholar,8Vincent M.E. Liu W. Haney E.B. Ismagilov R.F. Microfluidic stochastic confinement enhances analysis of rare cells by isolating cells and creating high density environments for control of diffusible signals.Chem Soc Rev. 2010; 39: 974-984Crossref PubMed Scopus (82) Google Scholar resulting in a reliable quantification of MRD in about 20% to 30% of these samples.9Drandi D. Kubiczkova-Besse L. Ferrero S. Dani N. Passera R. Mantoan B. Gambella M. Monitillo L. Saraci E. Ghione P. Genuardi E. Barbero D. Omedè P. Barberio D. Hajek R. Vitolo U. Palumbo A. Cortelazzo S. Boccadoro M. Inghirami G. Ladetto M. Minimal residual disease detection by droplet digital PCR in multiple myeloma, mantle cell lymphoma, and follicular lymphoma: a comparison with real-time PCR.J Mol Diagn. 2015; 17: 652-660Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 10Della Starza I. Nunes V. Cavalli M. De Novi L.A. Ilari C. Apicella V. Vitale A. Testi A.M. Del Giudice I. Chiaretti S. Foà R. Guarini A. Comparative analysis between RQ-PCR and digital-droplet-PCR of immunoglobulin/T-cell receptor gene rearrangements to monitor minimal residual disease in acute lymphoblastic leukaemia.Br J Haematol. 2016; 174: 541-549Crossref PubMed Scopus (45) Google Scholar, 11Cavalli M. De Novi L.A. Della Starza I. Cappelli L.V. Nunes V. Pulsoni A. Del Giudice I. Guarini A. Foà R. Comparative analysis between RQ-PCR and digital droplet PCR of BCL2/IGH gene rearrangement in the peripheral blood and bone marrow of early stage follicular lymphoma.Br J Haematol. 2017; 177: 588-596Crossref PubMed Scopus (30) Google Scholar Likewise, several groups have documented the value of NGS technologies for MRD detection in precursor and mature B-cell tumors.6Kotrova M. van der Velden V.H. van Dongen J.J. Formankova R. Sedlacek P. Brüggemann M. Zuna J. Stary J. Trka J. Fronkova E. Next-generation sequencing indicates false-positive MRD results and better predicts prognosis after SCT in patients with childhood ALL.Bone Marrow Transplant. 2017; 52: 962-968Crossref PubMed Scopus (57) Google Scholar,12Logan A.C. Zhang B. Narasimhan B. Carlton V. J. M. M.R. M. Minimal residual disease quantification and sequencing predicts relapse in 2013; PubMed Scopus Google M. Brüggemann M. Monitillo L. Ferrero S. D. Barbero D. Palumbo A. Passera R. Boccadoro M. M. N. J. Carlton V. H. M. C. Next-generation sequencing and real-time quantitative PCR for minimal residual disease detection in B-cell PubMed Scopus Google Scholar the NGS in ALL have that a level of A.C. Zhang B. Narasimhan B. Carlton V. J. M. M.R. M. Minimal residual disease quantification and sequencing predicts relapse in 2013; PubMed Scopus Google M. Brüggemann M. Monitillo L. Ferrero S. D. Barbero D. Palumbo A. Passera R. Boccadoro M. M. N. J. Carlton V. H. M. C. Next-generation sequencing and real-time quantitative PCR for minimal residual disease detection in B-cell PubMed Scopus Google Scholar is when of DNA are have that NGS may be more RQ-PCR in relapse in ALL patients after as as after stem cell M. van der Velden V.H. van Dongen J.J. Formankova R. Sedlacek P. Brüggemann M. Zuna J. Stary J. Trka J. Fronkova E. Next-generation sequencing indicates false-positive MRD results and better predicts prognosis after SCT in patients with childhood ALL.Bone Marrow Transplant. 2017; 52: 962-968Crossref PubMed Scopus (57) Google M. K. E. M. V. K. J. M. M. C. M. M. Stary J. Trka J. Fronkova E. The of next-generation sequencing MRD detection for relapse compared with in childhood 2015; PubMed Scopus Google Scholar analyzed by RQ-PCR and ddPCR 116 follow-up samples at four from 44 adult ALL patients enrolled in the GIMEMA LAL1913 protocol for R. Chiaretti S. E. L. E. P. G. D. A. M. De P. M. A. V. P. G. M. Della Starza I. A. L. De M.S. Guarini A. Vitale A. P. M. A. R. results of GIMEMA LAL1913 for adult patients with Philadelphia-negative acute lymphoblastic leukemia European Scholar to evaluate the potential of ddPCR to the MRD to the rate of quantification of low disease and to improve patients' risk stratification. The analysis was also by NGS and in a of ALL samples. A of 116 bone marrow samples from 44 adult ALL patients (35 B-lineage and 9 between and and enrolled in the GIMEMA LAL1913 were by RQ-PCR and ddPCR IG/TR gene rearrangements as molecular The analysis was by at four and and the was at at at and at Forty-seven of the 116 samples from on material were also by The follow-up of the analyzed was to clinical risk based on diagnostic and molecular was used at to and patients at R. Chiaretti S. E. L. E. P. G. D. A. M. De P. M. A. V. P. G. M. Della Starza I. A. L. De M.S. Guarini A. Vitale A. P. M. A. R. results of GIMEMA LAL1913 for adult patients with Philadelphia-negative acute lymphoblastic leukemia European Scholar patients chemotherapy, with MRD analysis at four and of to risk and patients with MRD at or or at were MRD and patients with MRD at and and at were MRD MRD results at and the time points for stem cell DNA samples were by PCR to and gene T. E. P. P. M. Panzer-Grumayer E.R. A. J.F. van Dongen J.J. and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia immunoglobulin and cell receptor gene rearrangements and as PCR of the of minimal residual disease in acute PubMed Scopus Google Scholar, Dongen J.J. M. M. E. E. R. van J. D. C. M. M. A. M. and of PCR and protocols for detection of immunoglobulin and T-cell receptor gene in of the 17: PubMed Scopus Google Scholar, T. van der Velden M. van E.R. van Dongen J.J.M. rearrangements are in lymphoblastic leukemia but rare in PubMed Scopus Google Scholar used as for MRD MRD RQ-PCR was and according to the EuroMRD as der Velden V.H. Cazzaniga G. Schrauder A. Hancock J. Bader P. Panzer-Grumayer E.R. Flohr T. Sutton R. Cave H. Madsen H.O. Cayuela J.M. Trka J. Eckert C. Foroni L. Zur Stadt U. Beldjord K. Raff T. van der Schoot C.E. van Dongen J.J.M. European Study Group on MRD detection in ALL (ESG-MRD-ALL)Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data.Leukemia. 2007; 21: 604-611Crossref PubMed Scopus (535) Google Scholar The MRD ddPCR and NGS were as M. van der Velden V.H. van Dongen J.J. Formankova R. Sedlacek P. Brüggemann M. Zuna J. Stary J. Trka J. Fronkova E. Next-generation sequencing indicates false-positive MRD results and better predicts prognosis after SCT in patients with childhood ALL.Bone Marrow Transplant. 2017; 52: 962-968Crossref PubMed Scopus (57) Google Starza I. De Novi L.A. A. D. W. Cavalli M. L. R. Apicella V. Ilari C. Vitale A. Testi A.M. Inghirami G. Chiaretti S. Foà R. Guarini A. Digital droplet PCR and next-generation sequencing refine minimal residual disease monitoring in acute lymphoblastic PubMed Scopus Google Scholar, M. J. M. Carlton P. E. D. for minimal residual disease detection in acute lymphoblastic 2012; 120: PubMed Scopus Google Scholar, Starza I. Nunes V. D. Cavalli M. A. M. De Novi L.A. R. C. L. A. Guarini A. V. A. G. Foà R. Cazzaniga G. digital PCR MRD risk definition in childhood B-cell precursor acute lymphoblastic PubMed Scopus Google Scholar for in were the or the levels by RQ-PCR or ddPCR were in survival was defined as the time from the of to or follow-up and the the was used to evaluate between were analyzed the Foundation for The in the the MRD monitoring within the GIMEMA MRD were on samples of adults by ALL enrolled in a GIMEMA The GIMEMA is LAL1913 number by the of the on and by all MRD monitoring was of the all patients the the the comparison of MRD results by RQ-PCR and ddPCR a concordance rate of 79% (P < for samples classified as positive quantifiable or (NEG) by results were in 21% of samples. were of which were and of which were in the samples, were and were the use of ddPCR significantly reduced the proportion of samples defined as of 116 versus of 116 (P = MRD in of RQ-PCR-PNQ samples and proved in of Moreover, ddPCR also allowed to quantify the disease in of samples that were RQ-PCR-NEG, MRD quantification by 14% in samples defined RQ-PCR-PNQ or and Comparison of RQ-PCR and ddPCR NGS MRD were on 116 samples at four time points and by RQ-PCR and ddPCR. 116 samples, were also analyzed by NGS are in droplet digital minimal residual next-generation positive not positive RQ-PCR, real-time quantitative in a were on 116 samples at four time points and by RQ-PCR and ddPCR. 116 samples, were also analyzed by NGS are in droplet digital minimal residual next-generation positive not positive RQ-PCR, real-time quantitative of the 116 samples analyzed by RQ-PCR and MRD was also analyzed by of samples resulted and of samples resulted NEG. The concordance rate between NGS and RQ-PCR and ddPCR was and NGS the disease in samples classified as PNQ or by RQ-PCR, the ddPCR results in of the in samples that were NGS was to quantify the disease with an MRD level of Moreover, in RQ-PCR-PNQ NGS was and in but RQ-PCR-NEG, NGS was of RQ-PCR versus NGS and ddPCR versus NGS MRD were on samples at four time points and between RQ-PCR and NGS and ddPCR and NGS are in droplet digital minimal residual next-generation positive not positive RQ-PCR, real-time quantitative in a were on samples at four time points and between RQ-PCR and NGS and ddPCR and NGS are in droplet digital minimal residual next-generation positive not positive RQ-PCR, real-time quantitative The RQ-PCR and ddPCR MRD analysis was at and for a of 116 samples At and and to the the RQ-PCR and ddPCR were on samples. By RQ-PCR, of were 9 of were and of were by of were of was and of were NEG. at these the results in of samples were were were and was A of of the samples was analyzed also by samples proved and were NEG. the NGS of samples were samples were and NGS resulted were and NGS was and were NGS was and that ddPCR and NGS results at the early At and and samples were analyzed by RQ-PCR and ddPCR. By RQ-PCR, resulted resulted and resulted by proved proved and proved NEG. The were in of samples and the samples, were also analyzed by and samples proved being samples that were resulted by and that was was by of 44 patients relapsed after a follow-up of to relapses in patients who were by RQ-PCR but proved by ddPCR NGS and in patients defined by all survival was on the basis of the patients' disease levels at a and At by of RQ-PCR, were no between = = and PNQ = At variance, by ddPCR resulted in the of PNQ to an in = and = cases. event-free survival was different between the (P = At and representing time points for patients' to patients with levels by RQ-PCR and ddPCR a significantly compared to with PNQ or no with the results observed at ddPCR more patients who a relapse of five at and three of five at compared to RQ-PCR of five at and three of five at patients were classified based on the possibility to quantify disease levels by in more to the of patients' over time and to with quantifiable disease in more the of patients with more were by RQ-PCR and were by ddPCR by ddPCR a significantly event-free survival by RQ-PCR was in four of five relapsed patients by ddPCR versus of five by RQ-PCR RQ-PCR MRD monitoring in a of samples with low MRD levels be the use of the EuroMRD guidelines.3van der Velden V.H. Cazzaniga G. Schrauder A. Hancock J. Bader P. Panzer-Grumayer E.R. Flohr T. Sutton R. Cave H. Madsen H.O. Cayuela J.M. Trka J. Eckert C. Foroni L. Zur Stadt U. Beldjord K. Raff T. van der Schoot C.E. van Dongen J.J.M. European Study Group on MRD detection in ALL (ESG-MRD-ALL)Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data.Leukemia. 2007; 21: 604-611Crossref PubMed Scopus (535) Google Scholar low disease levels are to the of the analytical methods, is to results, and a precise MRD definition in these cases. the 116 samples from 44 adult ALL patients enrolled in the GIMEMA LAL1913 protocol for ALL at four were analyzed by RQ-PCR and ddPCR to evaluate the potential of ddPCR to the MRD quantification of low disease levels and patients' risk stratification. The comparison of MRD results by RQ-PCR and ddPCR a concordance rate of 79% at the The of ddPCR allowed to quantifiable positive samples and to quantify the disease in 6.6% of samples defined as by RQ-PCR, MRD quantification of These the and of ddPCR that to an MRD also in low positive samples in that ddPCR can provide a more and precise stratification for cases with a to cases from defined as or PNQ by Moreover, the value of represents the most and At and and RQ-PCR and ddPCR were in samples, with by ddPCR. at and and RQ-PCR and ddPCR were in samples, and in ddPCR the MRD compared with RQ-PCR versus By survival ddPCR and RQ-PCR were to patients' relapse at and with an of ddPCR in more the number of has because the detection of levels of disease could to in clinical trials and potentially patients from the is to that no relapses were recorded in patients whose samples were defined as RQ-PCR-PNQ but proved by ddPCR. These in by Raff T. N. S. R. M. S. S. Horst H.A. M. Hoelzer D. Brüggemann M. Study Group relapse in adult ALL patients by MRD monitoring and after from the and 2007; 109: PubMed Scopus Google Scholar relapses were observed also in cases with MRD within the quantitative a of in the three relapses were documented in RQ-PCR-PNQ/NEG these three cases were by thus the of ddPCR over when the disease level multiple ddPCR was to the of is more precise or as the of ALL over time. all these with the of the the clinical of ddPCR over RQ-PCR in patients' stratification. a number of samples = MRD was also analyzed by which a concordance rate of with RQ-PCR and of with ddPCR. It confirmed the ddPCR quantification in samples classified as RQ-PCR-PNQ or and allowed to the rate of as positive and quantifiable three samples that resulted 21% versus versus are on increasing the number of cases to also by However, NGS in be more in MRD be that of DNA, not at follow-up and analysis by all three in samples from patients enrolled in GIMEMA trials cases from and from Starza I. De Novi L.A. A. D. W. Cavalli M. L. R. Apicella V. Ilari C. Vitale A. Testi A.M. Inghirami G. Chiaretti S. Foà R. Guarini A. Digital droplet PCR and next-generation sequencing refine minimal residual disease monitoring in acute lymphoblastic PubMed Scopus Google Scholar By increasing the number of patients and samples from a can the of ddPCR in the rate of quantification in low positive samples, with a concordance compared with NGS versus with these in a analysis between RQ-PCR and ddPCR in a in with the Starza I. Nunes V. D. Cavalli M. A. M. De Novi L.A. R. C. L. A. Guarini A. V. A. G. Foà R. Cazzaniga G. digital PCR MRD risk definition in childhood B-cell precursor acute lymphoblastic PubMed Scopus Google Scholar that within a of childhood ALL patients defined as early a high disease at and resulting RQ-PCR PNQ at most relapses in cases that proved MRD quantifiable by ddPCR at (P < the patients with a or PNQ ddPCR MRD at a better patients with a high MRD at and at and to that of patients enrolled in the these that ddPCR is a as as RQ-PCR in and MRD at all analyzed and more when the RQ-PCR quantitative is to and Moreover, ddPCR allows the quantitation of nucleic acid targets without the need of the thus not limiting the possibility of monitoring patients over time. have that ddPCR is an to monitor MRD in different to relapse by samples and to to a potential in patients' risk M. De Novi L.A. Della Starza I. Cappelli L.V. Nunes V. Pulsoni A. Del Giudice I. Guarini A. Foà R. Comparative analysis between RQ-PCR and digital droplet PCR of BCL2/IGH gene rearrangement in the peripheral blood and bone marrow of early stage follicular lymphoma.Br J Haematol. 2017; 177: 588-596Crossref PubMed Scopus (30) Google Starza I. Nunes V. D. Cavalli M. A. M. De Novi L.A. R. C. L. A. Guarini A. V. A. G. Foà R. Cazzaniga G. digital PCR MRD risk definition in childhood B-cell precursor acute lymphoblastic PubMed Scopus Google D. M. I. A. L. G. M. Genuardi E. Mantoan B. P. M. Starza E. R. C. M. C. Eckert C. V. Cortelazzo S. M. C. Ladetto M. Ferrero S. E. digital PCR quantification of mantle cell follow-up samples from four trials of the European PubMed Scopus Google N. G. L. A. G. Digital a reliable for and monitoring J Mol 21: Scopus Google Scholar The have the of a of and sensitivity, and the possibility of about the gene rearrangement of each At the use to monitor MRD in clinical protocols is by the lack of a to MRD in different clinical The EuroMRD Consortium is to The will be a analysis by for which more are of samples classified as PNQ by RQ-PCR at to to a of ALL patients' stratification and and analyzed the and the and and and clinical and the and and R.F. the and the

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