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Droplet Digital PCR Is a Robust Tool for Monitoring Minimal Residual Disease in Adult Philadelphia-Positive Acute Lymphoblastic Leukemia

2018; Elsevier BV; Volume: 20; Issue: 4 Linguagem: Inglês

10.1016/j.jmoldx.2018.03.002

ISSN

1943-7811

Autores

Nicoletta Coccaro, Luisa Anelli, Antonella Zagaria, Paola Casieri, Giuseppina Tota, Paola Orsini, Luciana Impera, Angela Minervini, Crescenzio Francesco Minervini, Cosimo Cumbo, Elisa Parciante, Paola Carluccio, Claudia Brunetti, Giorgina Specchia, Francesco Albano,

Tópico(s)

Chronic Lymphocytic Leukemia Research

Resumo

The breakpoint cluster region–abelson 1 p190 fusion transcript is the most frequent variant observed in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). Qualitative-PCR and real-time quantitative PCR are the currently used methods to monitor minimal residual disease (MRD) in Ph+ ALL patients; for the latter, full standardization and an international quality validation are lacking. Here, we developed a droplet digital PCR (ddPCR) assay for MRD monitoring in p190+ ALL cases. The analytical performance was assessed by the limit-of-detection determination, showing a reliability, sensitivity, and precision of the assay of up to 0.001%. Comparison of results obtained with qualitative PCR and ddPCR in 117 follow-up samples from 16 of 26 Ph+ ALL patients showed discordant results in 27% of cases (32 of 117). Real-time quantitative PCR analysis of 19 ddPCR-positive samples with a low tumor burden failed to provide quantitative results in 63% of cases (12 of 19). These results highlight that in p190+ ALL the ddPCR method has a sufficient analytical performance for very low MRD monitoring and for predicting molecular relapse several months before hematologic relapse. In conclusion, MRD monitoring by ddPCR may better stratify Ph+ ALL patients at risk of disease progression. The breakpoint cluster region–abelson 1 p190 fusion transcript is the most frequent variant observed in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). Qualitative-PCR and real-time quantitative PCR are the currently used methods to monitor minimal residual disease (MRD) in Ph+ ALL patients; for the latter, full standardization and an international quality validation are lacking. Here, we developed a droplet digital PCR (ddPCR) assay for MRD monitoring in p190+ ALL cases. The analytical performance was assessed by the limit-of-detection determination, showing a reliability, sensitivity, and precision of the assay of up to 0.001%. Comparison of results obtained with qualitative PCR and ddPCR in 117 follow-up samples from 16 of 26 Ph+ ALL patients showed discordant results in 27% of cases (32 of 117). Real-time quantitative PCR analysis of 19 ddPCR-positive samples with a low tumor burden failed to provide quantitative results in 63% of cases (12 of 19). These results highlight that in p190+ ALL the ddPCR method has a sufficient analytical performance for very low MRD monitoring and for predicting molecular relapse several months before hematologic relapse. In conclusion, MRD monitoring by ddPCR may better stratify Ph+ ALL patients at risk of disease progression. Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) accounts for 20% to 30% of adult cases of ALL.1Wetzler M. Dodge R.K. Mrózek K. Carroll A.J. Tantravahi R. Block A.W. Pettenati M.J. Le Beau M.M. Frankel S.R. Stewart C.C. Szatrowski T.P. Schiffer C.A. Larson R.A. Bloomfield C.D. Prospective karyotype analysis in adult acute lymphoblastic leukemia: the cancer and leukemia group B experience.Blood. 1999; 93: 3983-3993PubMed Google Scholar, 2Faderl S. Jeha S. Kantarjian H.M. The biology and therapy of adult acute lymphoblastic leukemia.Cancer. 2003; 98: 1337-1354Crossref PubMed Scopus (136) Google Scholar, 3Burmeister T. Schwartz S. Bartram C.R. Gokbuget N. Hoelzer D. Thiel E. GMALL study groupPatients' age and BCR-ABL frequency in adult B-precursor ALL: a retrospective analysis from the GMALL study group.Blood. 2008; 112: 918-919Crossref PubMed Scopus (108) Google Scholar Cytogenetic and molecular qualitative analyses by PCR usually are used to confirm the morphologic diagnosis of Ph+ ALL and to assess which transcript variant is generated in each Ph+ ALL case.4van 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. Diáz 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 (1011) Google Scholar The breakpoint cluster region–abelson (BCR-ABL) 1 p190 fusion transcript is the most frequent variant observed in 24% of ALL cases.3Burmeister T. Schwartz S. Bartram C.R. Gokbuget N. Hoelzer D. Thiel E. GMALL study groupPatients' age and BCR-ABL frequency in adult B-precursor ALL: a retrospective analysis from the GMALL study group.Blood. 2008; 112: 918-919Crossref PubMed Scopus (108) Google Scholar BCR-ABL1 tyrosine kinase inhibitors (TKIs) are considered an important component of treatment for adult patients affected by Ph+ ALL.5Fielding A.K. How I treat Philadelphia chromosome-positive acute lymphoblastic leukemia.Blood. 2010; 116: 3409-3417Crossref PubMed Scopus (73) Google Scholar, 6Liu-Dumlao T. Kantarjian H. Thomas D.A. O'Brien S. Ravandi F. Philadelphia-positive acute lymphoblastic leukemia: current treatment options.Curr Oncol Rep. 2012; 14: 387-394Crossref PubMed Scopus (65) Google Scholar, 7Malagola M. Papayannidis C. Baccarani M. Tyrosine kinase inhibitors in Ph+ acute lymphoblastic leukaemia: facts and perspectives.Ann Hematol. 2016; 95: 681-693Crossref PubMed Scopus (34) Google Scholar In fact, recent studies reported an improved overall outcome after treating Ph+ ALL patients with the combination of imatinib and multi-agent chemotherapy.8Lim S.N. Joo Y.D. Lee K.H. Kim D.Y. Lee J.H. Lee J.H. Chi H.S. Yun S.C. Lee W.S. Lee S.M. Park S. Kim I. Sohn S.K. Moon J.H. Ryoo H.M. Bae S.H. Hyun M.S. Kim M.K. Kim H.J. Yang D.H. Eom H.S. Lee G.W. Jung C.W. Won J.H. Kim H. Lee J.H. Shin H.J. Jang D.Y. Long-term follow-up of imatinib plus combination chemotherapy in patients with newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia.Am J Hematol. 2015; 90: 1013-1020Crossref PubMed Scopus (26) Google Scholar Before the introduction of TKI therapy, Ph+ ALL patients had a poor prognosis, and a better outcome occurred mainly only in patients who underwent allogeneic stem cell transplant (allo-SCT) in first complete remission.9Ravandi F. Kebriaei P. Philadelphia chromosome-positive acute lymphoblastic leukemia.Hematol Oncol Clin North Am. 2009; 23: 1043-1063Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar The investigation of residual BCR-ABL1 transcript levels in Ph+ ALL patients shortly after starting TKI or during treatment is a strong prognostic factor, and is useful to detect early relapse and indicate a rapid change of therapy before hematologic relapse.10Fielding A.K. Treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia in adults: a broader range of options, improved outcomes, and more therapeutic dilemmas.Am Soc Clin Oncol Educ Book. 2015; : e352-e359Crossref PubMed Scopus (30) Google Scholar, 11Foa R. Vitale A. Vignetti M. Meloni G. Guarini A. De Propris M.S. Elia L. Paoloni F. Fazi P. Cimino G. Nobile F. Ferrara F. Castagnola C. Sica S. Leoni P. Zuffa E. Fozza C. Luppi M. Candoni A. Iacobucci I. Soverini S. Mandelli F. Martinelli G. Baccarani M. GIMEMA Acute Leukemia Working Partydasatinib as first-line treatment for adult patients with Philadelphia chromosome-positive acute lymphoblastic leukemia.Blood. 2011; 118: 6521-6528Crossref PubMed Scopus (338) Google Scholar, 12Ravandi F. Jorgensen J.L. Thomas D.A. O'Brien S. Garris R. Faderl S. Huang X. Wen S. Burger J.A. Ferrajoli A. Kebriaei P. Champlin R.E. Estrov Z. Challagundla P. Wang S.A. Luthra R. Cortes J.E. Kantarjian H.M. Detection of MRD may predict the outcome of patients with Philadelphia chromosome-positive ALL treated with tyrosine kinase inhibitors plus chemotherapy.Blood. 2013; 122: 1214-1221Crossref PubMed Scopus (154) Google Scholar, 13Short N.J. Jabbour E. Sasaki K. Patel K. O'Brien S.M. Cortes J.E. Garris R. Issa G.C. Garcia-Manero G. Luthra R. Thomas D. Kantarjian H. Ravandi F. Impact of complete molecular response on survival in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia.Blood. 2016; 128: 504-507Crossref PubMed Scopus (142) Google Scholar, 14Yoon J.H. Yhim H.Y. Kwak J.Y. Ahn J.S. Yang D.H. Lee J.J. Kim S.J. Kim J.S. Park S.J. Choi C.W. Eom H.S. Park S.K. Choi S.Y. Kim S.H. Kim D.W. Lee S. Minimal residual disease-based effect and long-term outcome of first-line dasatinib combined with chemotherapy for adult Philadelphia chromosome-positive acute lymphoblastic leukemia.Ann Oncol. 2016; 27: 1081-1088Crossref PubMed Scopus (40) Google Scholar, 15Chiaretti S. Vitale A. Vignetti M. Piciocchi A. Fazi P. Elia L. Falini B. Ronco F. Ferrara F. De Fabritiis P. Luppi M. La Nasa G. Tedeschi A. Califano C. Fanin R. Dore F. Mandelli F. Meloni G. Foà R. A sequential approach with imatinib, chemotherapy and transplant for adult Ph+ acute lymphoblastic leukemia: final results of the GIMEMA LAL 0904 study.Haematologica. 2016; 101: 1544-1552Crossref PubMed Scopus (54) Google Scholar To date, minimal residual disease (MRD) monitoring of the p190 fusion transcript in Ph+ ALL patients has been performed by qualitative PCR (QL-PCR) or by real-time quantitative PCR (qPCR); the latter is a sensitive, easy-to-perform method, but lacks full standardization and international quality validation.16Lee S. Kim D.W. Cho B. Kim Y.J. Kim Y.L. Hwang J.Y. Park Y.H. Shin H.J. Park C.Y. Min W.S. Kim H.K. Kim C.C. Risk factors for adults with Philadelphia-chromosome-positive acute lymphoblastic leukaemia in remission treated with allogeneic bone marrow transplantation: the potential of real-time quantitative reverse-transcription polymerase chain reaction.Br J Haematol. 2003; 120: 145-153Crossref PubMed Scopus (52) Google Scholar, 17Lee S. Kim D.W. Cho B.S. Yoon J.H. Shin S.H. Yahng S.A. Lee S.E. Eom K.S. Kim Y.J. Chung N.G. Kim H.J. Min C.K. Lee J.W. Min W.S. Park C.W. Impact of minimal residual disease kinetics during imatinib-based treatment on transplantation outcome in Philadelphia chromosome-positive acute lymphoblastic leukemia.Leukemia. 2012; 26: 2367-2374Crossref PubMed Scopus (71) Google Scholar In this study, we performed a quantitative analysis of the BCR-ABL1 p190 fusion transcript in 26 Ph+ ALL patients at diagnosis and during MRD monitoring at several follow-up time points by droplet digital PCR (ddPCR).18Baker M. Digital PCR hits its stride.Nat Methods. 2012; 9: 541-544Crossref Scopus (384) Google Scholar, 19Hindson 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 (945) Google Scholar The ddPCR performance was compared with QL-PCR and qPCR, proving that the ddPCR test is a highly sensitive method for monitoring BCR-ABL1 transcript levels in Ph+ ALL cases, showing a remarkable reliability, sensitivity, and precision. The study included 26 patients (median age, 50 years; minimum, 24 years; maximum, 74 years; 10 men and 16 women) with newly diagnosed Ph+ ALL. The morphologic diagnosis was confirmed in all cases by QL-PCR, karyotypic analysis, and/or fluorescence in situ hybridization, as previously reported (Table 1).20Albano F. Anelli L. Zagaria A. Archidiacono N. Liso V. Specchia G. Rocchi M. "Home-brew" FISH assay shows higher efficiency than BCR-ABL dual color, dual fusion probe in detecting microdeletions and complex rearrangements associated with t(9;22) in chronic myeloid leukemia.Cancer Genet Cytogenet. 2007; 174: 121-126Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 21Albano F. Anelli L. Zagaria A. Coccaro N. Casieri P. Rossi A.R. Vicari L. Liso V. Rocchi M. Specchia G. Non random distribution of genomic features in breakpoint regions involved in chronic myeloid leukemia cases with variant t(9;22) or additional chromosomal rearrangements.Mol Cancer. 2010; 9: 120Crossref PubMed Scopus (28) Google Scholar, 22Shaffer L.G. McGowan-Jordan J. Schmid M. ISCN 2013: An International System for Human Cytogenetic Nomenclature. Karger, Basel, Switzerland2013Google Scholar The median follow-up time of the entire cohort was 2.6 years (minimum, 4 months; maximum, 14 years). The main characteristics of the patients are reported in Table 1.Table 1The Main Characteristics of the Ph+ ALL PatientsCaseAge/sexWBC (×109/L)Cytogenetic analysis22Shaffer L.G. McGowan-Jordan J. Schmid M. ISCN 2013: An International System for Human Cytogenetic Nomenclature. Karger, Basel, Switzerland2013Google ScholarFISH analysis22Shaffer L.G. McGowan-Jordan J. Schmid M. ISCN 2013: An International System for Human Cytogenetic Nomenclature. Karger, Basel, Switzerland2013Google ScholarQL-PCR4van 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. Diáz 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 (1011) Google Scholar124/F17346,XX,t(9; 22) (q34; q11)[20]NAp190 BCR-ABL1264/M14.92NPnuc ish(ABL1,BCR) × 3(ABL con BCRx2)[400]p190 BCR-ABL1361/F5.97NAish t(9;22)(ABL1+,BCR+;BCR+,ABL1+)[20]p190 BCR-ABL1462/M303NPnuc ish(ABL1,BCR) × 3(ABL con BCRx2)[400]p190 BCR-ABL1533/F66NAnuc ish(ABL1,BCR) × 3(ABL con BCRx2)[400]p190 BCR-ABL1650/F78.646,XX,der(9)t(2; 9) (p?; p?)t(9; 12; 22) (q34; p?13; q11)[4]/49,XX,+der(2)t(2; 9) (p?; p?),+8,der(9)t(2; 9) (p?; p?)t(9; 12; 22) (q34; p?13; q11),+22[16]nuc ish(ABL1,BCR) × 5(ABL con BCRx4)[400]p190 BCR-ABL1741/M24.546,XY,del(9p),t(9; 22) (q34; q11)[16]/46,XY[4]NAp190 BCR-ABL1869/F3.2645–46,XX,add(2) (q37),der(5q),−7,t(9; 22) (q34; q11),12p−,-?14,?der(21),+?Ph[cp19]/46,XX[1]ish t(9; 22) (ABL+,BCR+;BCR+,ABL1+)der(22) (BCR+,ABL1+)[18/20]p190 BCR-ABL1951/F2647,XX,t(2; 12) (q?31; q?22),t(9; 22) (q34; q11),+der22 t(9; 22) (q34; q11)[8]/94,XXXX,t(2; 12) (q?31; q?32) × 2,t(9; 22) (q34; q11) × 2,+der(22)t(9; 22) (q34; q11) × 2[4]/46,XX[8]ish t(9; 22) (ABL+,BCR+;BCR+,ABL1+)der(22) (BCR+,ABL1+)[9/20]/t(9; 22) (ABL+,BCR+;BCR+,ABL1+) × 2der(22) (BCR+,ABL1+) × 2[3/20]p190 BCR-ABL11074/F2.1NANAp190 BCR-ABL11130/F8.4946,XX,t(9; 22) (q34; q11)[14]/46,XX[6]NAp190 BCR-ABL11259/F2NAish t(9; 22) (ABL1+,BCR+;BCR+,ABL1+)[20]p190 BCR-ABL11343/F20.7NAish t(9; 22) (ABL1+,BCR+;BCR+,ABL1+)[20]p190 BCR-ABL11432/M42.9NANAp190 BCR-ABL11542/M17.3NANAp190 BCR-ABL11671/M16.43NANAp190 BCR-ABL11732/M4.52NANAp190 BCR-ABL11845/F4.6NAish t(9; 22) (ABL1+,BCR+;BCR+,ABL1+)[20]p190 BCR-ABL11942/M29.3NANAp190 BCR-ABL12027/F27.45NANAp190 BCR-ABL12168/F9.79NANAp190 BCR-ABL12269/F8NANAp190 BCR-ABL12329/M2.6NPnuc ish(ABL1,BCR) × 3(ABL con BCRx2)[400]p190 BCR-ABL12465/F23.144,XX,−8,t(9; 22) (q34; q11),−15,?add(20q)[20]NAp190 BCR-ABL12558/M113.644,XY,−8,t(9; 22) (q34; q11),−11,−14,der(16)t(8; 16) (q?11.2; q?12),add(17p),der(20)t(14; 20) (q?11.2; q?11.2),+mar[10]/46,XY[10]NAp190 BCR-ABL12650/F3.8345,XX,−7,t(9; 22) (q34; q11)[20]NAp190 BCR-ABL1F, female; M, male; ALL, acute lymphoblastic leukemia; FISH, fluorescence in situ hybridization; ish, FISH on metaphases; NA, not available; NP, not performed because of the sample quality; nuc ish, FISH on interphase nuclei; Ph+, Philadelphia-positive; QL-PCR, qualitative PCR; WBC, white blood cell. Open table in a new tab F, female; M, male; ALL, acute lymphoblastic leukemia; FISH, fluorescence in situ hybridization; ish, FISH on metaphases; NA, not available; NP, not performed because of the sample quality; nuc ish, FISH on interphase nuclei; Ph+, Philadelphia-positive; QL-PCR, qualitative PCR; WBC, white blood cell. Total RNA was extracted from bone marrow samples at diagnosis or during the follow-up evaluation using the QIAamp RNA Blood Kit (catalog number 52304; Qiagen, Valencia, CA). The RNA concentration and purity were assessed using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). For qualitative and quantitative real-time experiments, 1000 ng of RNA samples were reverse transcribed into cDNA using the standardized Europe Against Cancer Reverse Transcription protocol.23Gabert J. Beillard E. van der Velden V.H. Bi W. Grimwade D. Pallisgaard N. Barbany G. Cazzaniga G. Cayuela J.M. Cavé H. Pane F. Aerts J.L. De Micheli D. Thirion X. Pradel V. González 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 (1231) Google Scholar For ddPCR experiments the iScript Advanced cDNA synthesis kit (Bio-Rad, Hercules, CA) was used; this kit offers a wide dynamic range for reverse transcription (from 100 fg to 7.5 μg of RNA). The input RNA amount was optimized on the estimated BCR-ABL1 abundance and sample availability: 1000 and 7500 ng of RNA were reverse transcribed for the samples at the onset and follow-up evaluation, respectively. Clinical samples at diagnosis from three patients and normal RNA samples from four healthy volunteers were mixed to generate a single sample called a p190-pool and diluent-pool, respectively. Identification of the BCR-ABL1 fusion gene and of the e1a2 fusion transcript, corresponding to the p190 chimeric protein, was performed at diagnosis on bone marrow samples from all 26 ALL patients according to the BIOMED-1 protocol.4van 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. Diáz 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 (1011) Google Scholar Samples at diagnosis were analyzed by a first round of PCR experiments with a sensitivity of approximately 10–3, whereas a second round of amplification (nested PCR) with internal primers was performed on follow-up samples (sensitivity, 10–3/10–4).4van 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. Diáz 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 (1011) Google Scholar The good quality of the cDNA sample was checked by verifying amplification of the housekeeping gene ACTB. Qualitative results were classified as first positive, nested positive, or nested negative. qPCR was performed on a LightCycler II 480 system (Roche Diagnostics, Monza, Italy), with the Ipsogen BCR-ABL1 p190 fusion transcript IVD kit (Qiagen). Absolute quantification was based on a standard curve built on five plasmid 10-fold dilutions (101, 102, 103, 104, and 105 copies) for the BCR-ABL1 p190 fusion transcript, and on three plasmid standard dilutions (103, 104, and 105 copies) for the ABL1 control gene. qPCR analysis was conducted starting from 100 ng of cDNA in triplicates for the fusion gene and in duplicates for the control gene. Results were interpreted according to the manufacturer's instructions for samples positive in all replicates and with a BCR-ABL1/ABL1 ratio >0.1; in particular, samples with an ABL1 copy number >1318 were considered acceptable for quantitative analysis. For samples with an undetectable BCR-ABL1 transcript, a minimum number of 10,000 copies of the ABL1 control gene for each replicate was required to classify the result as negative.24van 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. 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 (552) Google Scholar, 25Foroni L. Wilson G. Gerrard G. Mason J. Grimwade D. White H.E. de Castro D.G. Austin S. Awan A. Burt E. Clench T. Farruggia J. Hancock J. Irvine A.E. Kizilors A. Langabeer S. Milner B.J. Nickless G. Schuh A. Sproul A. Wang L. Wickham C. Cross N.C. Guidelines for the measurement of BCR-ABL1 transcripts in chronic myeloid leukaemia.Br J Haematol. 2011; 153: 179-190Crossref PubMed Scopus (80) Google Scholar Samples with a Cq higher than that of the lowest plasmid standard or with only one positive replicate were considered positive nonquantifiable (PNQ).24van 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. 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 (552) Google Scholar, 25Foroni L. Wilson G. Gerrard G. Mason J. Grimwade D. White H.E. de Castro D.G. Austin S. Awan A. Burt E. Clench T. Farruggia J. Hancock J. Irvine A.E. Kizilors A. Langabeer S. Milner B.J. Nickless G. Schuh A. Sproul A. Wang L. Wickham C. Cross N.C. Guidelines for the measurement of BCR-ABL1 transcripts in chronic myeloid leukaemia.Br J Haematol. 2011; 153: 179-190Crossref PubMed Scopus (80) Google Scholar ddPCR experiments were performed using primers and probes for BCR-ABL1 p190 as previously described and used in qPCR.23Gabert J. Beillard E. van der Velden V.H. Bi W. Grimwade D. Pallisgaard N. Barbany G. Cazzaniga G. Cayuela J.M. Cavé H. Pane F. Aerts J.L. De Micheli D. Thirion X. Pradel V. González 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 (1231) Google Scholar Glucuronidase beta (GUSB) was used as a control gene to confirm the good quality of cDNA samples. Both BCR-ABL1 p190 and GUSB primer and probes were used at a final concentration of 900 and 250 nmol/L and were labeled with the FAM and HEX reporters, respectively. Each experiment included a negative sample (from a healthy donor) and a positive control sample (previously tested diagnostic specimen), as well as a no-template control sample. Results were analyzed when the number of accepted droplets per well was at least 10,000 and the housekeeping gene provided correct (≥24,000 GUSB transcripts25Foroni L. Wilson G. Gerrard G. Mason J. Grimwade D. White H.E. de Castro D.G. Austin S. Awan A. Burt E. Clench T. Farruggia J. Hancock J. Irvine A.E. Kizilors A. Langabeer S. Milner B.J. Nickless G. Schuh A. Sproul A. Wang L. Wickham C. Cross N.C. Guidelines for the measurement of BCR-ABL1 transcripts in chronic myeloid leukaemia.Br J Haematol. 2011; 153: 179-190Crossref PubMed Scopus (80) Google Scholar, 26Cross N.C. White H.E. Colomer D. Ehrencrona H. Foroni L. Gottardi E. Lange T. Lion T. Machova Polakova K. Dulucq S. Martinelli G. Oppliger Leibundgut E. Pallisgaard N. Barbany G. Sacha T. Talmaci R. Izzo B. Saglio G. Pane F. Müller M.C. Hochhaus A. Laboratory recommendations for scoring deep molecular responses following treatment for chronic myeloid leukemia.Leukemia. 2015; 29: 999-1003Crossref PubMed Scopus (237) Google Scholar) and reproducible amplification. To define the limit of detection for the BCR-ABL1 p190 assay, the p190-pool was diluted serially into the diluent-pool. A 10-fold serial dilution was performed with the following concentrations: 100, 10–1, 10–2, 10–3, 10–4, and 10–5. The diluent-pool also was used as negative control. A total of 6 replicates using 750 ng of cDNA template were performed for each point of the serial dilution except for the lowest concentration (10–5; 0.001%), which was tested 9 times, and the negative sample, which was tested in 21 replicates.27Jennings L.J. George D. Czech J. Yu M. Joseph L. Detection and quantification of BCR-ABL1 fusion transcripts by droplet digital PCR.J Mol Diagn. 2014; 16: 174-179Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar BCR-ABL1 expression analysis by ddPCR was performed in 26 Ph+ ALL patients at disease onset and in 16 of 26 patients during the follow-up evaluation. The BCR-ABL1 and GUSB transcripts were analyzed in multiplex in the same well for the samples at diagnosis; by contrast, for the follow-up samples, the two genes were tested separately in different wells because different quantities of cDNA were needed. In follow-up samples, the analysis was performed on a larger quantity of cDNA to allow the detection of rare copies of the BCR-ABL1 fusion transcript. ddPCR experiments were performed using the QX-200 instrument (Bio-Rad); this system combines water–oil emulsion droplet technology with microfluidics. Each sample is partitioned into approximately 20,000 droplets by a droplet generator and each droplet is amplified by PCR. Then, droplets are streamed in single file on a droplet reader, which counts the fluorescent-positive and -negative droplets to define the target concentration. Fifty nanograms of cDNA template were used for the samples at the onset, and 750 ng were used for follow-up samples in a final volume of 20 μL. To increase the sensitivity, three replicates were run for follow-up samples; the GUSB transcript was run in a separate well using 100 ng of template. The 20-μL ddPCR reaction mixture then was loaded into the Bio-Rad DG8 droplet generator cartridge. A volume of 70 μL droplet generation oil was loaded for each sample. The cartridge was placed in the QX200 droplet generator. The generated droplets were transferred to an Eppendorf 96-well PCR plate (Eppendorf, Hamburg, Germany). The plate was sealed with a Bio-Rad pierceable foil heat seal, and samples were amplified on the T100 Bio-Rad thermal cycler. Thermal-cycling conditions were 95°C for 10 minutes (1 cycle), 94°C for 30 seconds (ramp rate, 2°C/second; 40 cycles), 60°C for 1 minute (ramp rate, 2°C/second; 40 cycles), 98°C for 10 minutes (1 cycle), and a 4°C hold. After amplification, the 96-well PCR plate was loaded on the Bio-Rad QX200 droplet reader and ddPCR data were analyzed with QuantaSoft analysis software version 1.7.4 (Bio-Rad). The target concentration in each sample was expressed as BCR-ABL1 copies/μg. The analytical performance of the BCR-ABL1 p190 ddPCR assay was defined by the limit-of-detection determination, using a pool of samples from three ALL patients at diagnosis, p190-pool (cases 7, 20, and 22), and a diluent pool of samples from healthy controls. Data from serial dilutions showed remarkable linearity, reliability, and a precision of up to 0.001% (Figure 1 and Table 2). Because the negative samples showed no background, even a single detected droplet was defined as significant.Table 2Analytical Performance Parameters of the ddPCR p190 AssayNumber of replicatesDilution series target value (%)Data mean (copies/μg)SD4100 (100)144,3003897610−1 (10)14,056482610−2 (1)131533610−3 (0.1)15219610−4 (0.01)17.43.5910−5 (0.001)3.81.921000cDNAs from three pretreatment samples were pooled (p190-pool) and diluted in total cDNA from healthy donors (diluent-pool).ddPCR, droplet digital PCR. Open table in a new tab cDNAs from three pretreatment samples were pooled (p190-pool) and diluted in total cDNA from healthy donors (diluent-pool). ddPCR, droplet digital PCR. Overall, all of the 26 Ph+ ALL at the onset and 253 follow-up samples were evaluable by QL-PCR and were classified as first positive (ie, positive with a first round of PCR: 95 of 279; 34%), nested positive (ie, positive with two rounds of PCR: 76 of 279; 27%), or nested negative (108 of 279; 39%). ddPCR experiments were performed successfully in all 26 ALL cases at disease onset; during follow-up monitoring, a total of 117 follow-up time points were analyzed by ddPCR in 16 of 26 patients (Supplemental Figure S1). Among the 47 first/nested positive samples, the ddPCR analysis showed concordant positive results in 46 cases (98%); in detail, the only discordant result was obtained for a nested positive sample that had a ddPCR-negative result (Figure 2). In comparison, of the 70 nested negative follow-up points, 39 (56%) also had negative results with ddPCR analysis (Figure 2). Follow-up points that were negative in ddPCR remained negative even when the experiments were repeated. For positive samples, quantitative results remained the same when the experiment was repeated and when the cDNA amount of three wells was loaded into a single well. Of note, the results also were reliable for positive samples with a very low copy number (<300 copies/μg). The amount of BCR-ABL1 fusion transcript at disease onset was very heterogeneous, and results showed a median concentration of 101 × 103 BCR-ABL1 copies/μg (minimum, 3.24 × 103 copies/μg; maximum, 1744 × 103 copies/μg). Of note, ALL patients who presented with

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