NPM1 for MRD? Droplet Like It's Hot!
2017; Elsevier BV; Volume: 19; Issue: 4 Linguagem: Inglês
10.1016/j.jmoldx.2017.04.008
ISSN1943-7811
Autores Tópico(s)Cancer Genomics and Diagnostics
ResumoCME 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 myeloid leukemia (AML) is a genetically heterogeneous neoplasm in which multiple distinct combinations of mutational events can give rise to leukemic blasts with similar morphologic and immunophenotypic characteristics.1Papaemmanuil E. Gerstung M. Bullinger L. Gaidzik V.I. Paschka P. Roberts N.D. Potter N.E. Heuser M. Thol F. Bolli N. Gundem G. Van Loo P. Martincorena I. Ganly P. Mudie L. McLaren S. O'Meara S. Raine K. Jones D.R. Teague J.W. Butler A.P. Greaves M.F. Ganser A. Dohner K. Schlenk R.F. Dohner H. Campbell P.J. Genomic classification and prognosis in acute myeloid leukemia.N Engl J Med. 2016; 374: 2209-2221Crossref PubMed Scopus (2261) Google Scholar, 2Metzeler K.H. Herold T. Rothenberg-Thurley M. Amler S. Sauerland M.C. Gorlich D. Schneider S. Konstandin N.P. Dufour A. Braundl K. Ksienzyk B. Zellmeier E. Hartmann L. Greif P.A. Fiegl M. Subklewe M. Bohlander S.K. Krug U. Faldum A. Berdel W.E. Wormann B. Buchner T. Hiddemann W. Braess J. Spiekermann K. AMLCG Study GroupSpectrum and prognostic relevance of driver gene mutations in acute myeloid leukemia.Blood. 2016; 128: 686-698Crossref PubMed Scopus (345) Google Scholar, 3Swerdlow S.H. Campo E. Harris N.L. Jaffe E.S. Pileri S.A. Stein H. Thiele J. Vardiman J. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue. International Agency for Research on Cancer, Lyon2008Google Scholar The underlying genetic diversity of AML is reflected in varying clinical courses of patients with this disease; some patients respond well to conventional chemotherapy and experience durable remissions, whereas others require bone marrow transplantation; however, some others succumb rapidly to their disease. Not only is AML genetically diverse across the patient population, but also AML cells within a single patient may be extensively heterogeneous.4Paguirigan A.L. Smith J. Meshinchi S. Carroll M. Maley C. Radich J.P. Single-cell genotyping demonstrates complex clonal diversity in acute myeloid leukemia.Sci Transl Med. 2015; 7: 281re2Crossref PubMed Scopus (104) Google Scholar A parental AML clone can give rise to multiple daughter subclones, each with a unique mutational profile and unique sensitivity to therapeutic interventions. Some of these daughter subclones may be undetectable at diagnosis but ultimately comprise most cells during relapse and disease progression.5Welch J.S. Ley T.J. Link D.C. Miller C.A. Larson D.E. Koboldt D.C. et al.The origin and evolution of mutations in acute myeloid leukemia.Cell. 2012; 150: 264-278Abstract Full Text Full Text PDF PubMed Scopus (1177) Google Scholar The wide spectrum of interpatient and intrapatient mutations in AML complicates assessment of patient prognosis at initial diagnosis. In fact, despite extensive mutational profiling by next-generation sequencing, predicting patient outcome is only marginally better than in preceding decades, with leukemic cell karyotype and patient age as the variables that most significantly correlate with patient survival.1Papaemmanuil E. Gerstung M. Bullinger L. Gaidzik V.I. Paschka P. Roberts N.D. Potter N.E. Heuser M. Thol F. Bolli N. Gundem G. Van Loo P. Martincorena I. Ganly P. Mudie L. McLaren S. O'Meara S. Raine K. Jones D.R. Teague J.W. Butler A.P. Greaves M.F. Ganser A. Dohner K. Schlenk R.F. Dohner H. Campbell P.J. Genomic classification and prognosis in acute myeloid leukemia.N Engl J Med. 2016; 374: 2209-2221Crossref PubMed Scopus (2261) Google Scholar In this issue of the Journal of Molecular Diagnostics, Mencia-Trinchant et al6Mencia-Trinchant N. Hu Y. Alas M.A. Ali F. Wouters B.J. Lee S. Ritchie E.K. Desai P. Guzman M.L. Roboz G.J. Hassane D.C. Minimal residual disease monitoring of acute myeloid leukemia by massively multiplex digital PCR in patients with NPM1 mutations.J Mol Diagn. 2017; 19: 537-548Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar characterize a novel clinical assay based on multiplex droplet digital PCR (ddPCR) to detect minimal residual disease (MRD) in the subset of patients with AML whose leukemia possesses mutations in NPM1. Although accurate outcome prediction for patients with AML before therapeutic intervention remains frustratingly difficult, the assessment of the actual response to therapy through determination of residual disease has emerged as a remarkably robust predictor of long-term outcome in these patients. Multiple studies have definitively found that the presence of MRD after therapy strongly predicts early relapse and relatively short survival.7Ivey A. Hills R.K. Simpson M.A. Jovanovic J.V. Gilkes A. Grech A. Patel Y. Bhudia N. Farah H. Mason J. Wall K. Akiki S. Griffiths M. Solomon E. McCaughan F. Linch D.C. Gale R.E. Vyas P. Freeman S.D. Russell N. Burnett A.K. Grimwade D. Assessment of minimal residual disease in standard-risk AML.N Engl J Med. 2016; 374: 422-433Crossref PubMed Scopus (512) Google Scholar, 8Gorello P. Cazzaniga G. Alberti F. Dell'Oro M.G. Gottardi E. Specchia G. Roti G. Rosati R. Martelli M.F. Diverio D. Lo Coco F. Biondi A. Saglio G. Mecucci C. Falini B. Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations.Leukemia. 2006; 20: 1103-1108Crossref PubMed Scopus (236) Google Scholar, 9Loken M.R. Alonzo T.A. Pardo L. Gerbing R.B. Raimondi S.C. Hirsch B.A. Ho P.A. Franklin J. Cooper T.M. Gamis A.S. Meshinchi S. Residual disease detected by multidimensional flow cytometry signifies high relapse risk in patients with de novo acute myeloid leukemia: a report from Children's Oncology Group.Blood. 2012; 120: 1581-1588Crossref PubMed Scopus (210) Google Scholar On the basis of these studies, recent guidelines for management of patients with AML require MRD assessment.10Dohner H. Estey E. Grimwade D. Amadori S. Appelbaum F.R. Buchner T. Dombret H. Ebert B.L. Fenaux P. Larson R.A. Levine R.L. Lo-Coco F. Naoe T. Niederwieser D. Ossenkoppele G.J. Sanz M. Sierra J. Tallman M.S. Tien H.F. Wei A.H. Lowenberg B. Bloomfield C.D. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel.Blood. 2017; 129: 424-447Crossref PubMed Scopus (3289) Google Scholar Multiple methods for MRD assessment have been developed, and clinical laboratories wishing to perform MRD analysis for AML must decide on an optimal platform. Current methods for determination of MRD in AML involve detection of genotypic aberrations that are specific to leukemic blasts or analysis of phenotypic differences that distinguish leukemic from normal cells.11Sung P.J. Luger S.M. Minimal residual disease in acute myeloid leukemia.Curr Treat Options Oncol. 2017; 18: 1Crossref PubMed Scopus (10) Google Scholar Although MRD assessment by any of these various techniques improves outcome prediction, all have important limitations. Genetic assays for AML cells, which typically rely on remarkably sensitive mutation-specific PCR, are hampered in their utility by the mutational variability across AML samples. For instance, translocation-specific real-time PCR has been used to determine MRD in patients with acute promyelocytic leukemia (ie, AML with PML-RARA fusions) and in patients with AML with RUNX1-RUNX1TI1 or CBFB-MYH11 fusions.12Chendamarai E. Balasubramanian P. George B. Viswabandya A. Abraham A. Ahmed R. Alex A.A. Ganesan S. Lakshmi K.M. Sitaram U. Nair S.C. Chandy M. Janet N.B. Srivastava V.M. Srivastava A. Mathews V. Role of minimal residual disease monitoring in acute promyelocytic leukemia treated with arsenic trioxide in frontline therapy.Blood. 2012; 119: 3413-3419Crossref PubMed Scopus (42) Google Scholar, 13Yin J.A. O'Brien M.A. Hills R.K. Daly S.B. Wheatley K. Burnett A.K. Minimal residual disease monitoring by quantitative RT-PCR in core binding factor AML allows risk stratification and predicts relapse: results of the United Kingdom MRC AML-15 trial.Blood. 2012; 120: 2826-2835Crossref PubMed Scopus (296) Google Scholar For these patients, MRD based on these assays has been correlated with disease recurrence and survival. Unfortunately, these specific translocations are only present in restricted subsets of patients with AML, and the assays are obviously ineffective for most of the remaining cases. Alternative nucleic acid–based assessment of MRD involves detection of transcripts that are expressed at relatively high levels in leukemic blasts yet are not expressed or expressed at low levels in normal cells. An example of a method based on RNA expression is quantitative RT-PCR for WT1 RNA.14Nowakowska-Kopera A. Sacha T. Florek I. Zawada M. Czekalska S. Skotnicki A.B. Wilms' tumor gene 1 expression analysis by real-time quantitative polymerase chain reaction for monitoring of minimal residual disease in acute leukemia.Leuk Lymphoma. 2009; 50: 1326-1332Crossref PubMed Scopus (23) Google Scholar Because most patients with AML have disease in which WT1 is overexpressed, this MRD assay is more widely applicable to the population of patients with AML than translocation-specific PCR assay. Unfortunately, analytic sensitivity, the sine qua non of MRD assays, is lower for the WT1 RNA than PCR based on specific mutations because normal hematopoietic elements express low levels of WT1 RNA.8Gorello P. Cazzaniga G. Alberti F. Dell'Oro M.G. Gottardi E. Specchia G. Roti G. Rosati R. Martelli M.F. Diverio D. Lo Coco F. Biondi A. Saglio G. Mecucci C. Falini B. Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations.Leukemia. 2006; 20: 1103-1108Crossref PubMed Scopus (236) Google Scholar The assay, therefore, can be used to follow up patients with AML over time for whom increasing levels of WT1 reflect increasing blast levels (and impending cytologic relapse), but it is less useful for single-time-point minimal disease detection. Multiparametric flow cytometry has also been extensively used for MRD detection in AML and numerous other hematologic malignant neoplasms.9Loken M.R. Alonzo T.A. Pardo L. Gerbing R.B. Raimondi S.C. Hirsch B.A. Ho P.A. Franklin J. Cooper T.M. Gamis A.S. Meshinchi S. Residual disease detected by multidimensional flow cytometry signifies high relapse risk in patients with de novo acute myeloid leukemia: a report from Children's Oncology Group.Blood. 2012; 120: 1581-1588Crossref PubMed Scopus (210) Google Scholar, 15Borowitz M.J. Wood B.L. Devidas M. Loh M.L. Raetz E.A. Salzer W.L. Nachman J.B. Carroll A.J. Heerema N.A. Gastier-Foster J.M. Willman C.L. Dai Y. Winick N.J. Hunger S.P. Carroll W.L. Larsen E. Prognostic significance of minimal residual disease in high risk B-ALL: a report from Children's Oncology Group study AALL0232.Blood. 2015; 126: 964-971Crossref PubMed Scopus (227) Google Scholar, 16Rawstron A.C. Fazi C. Agathangelidis A. Villamor N. Letestu R. Nomdedeu J. et al.A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study.Leukemia. 2016; 30: 929-936Crossref PubMed Scopus (154) Google Scholar Flow-based MRD detection in AML relies on restricted and reproducible antigen expression patterns of physiologically developing myeloid elements and the deviations from these patterns that leukemic cells typically exhibit. Blasts in most AML cases have abnormal expression of one or multiple immunophenotypic markers, so these MRD assays can be performed on samples from most patients with AML. Furthermore, cells are assessed individually and are, therefore, not susceptible to dilutional effects from nonleukemic cells that plague assays based on bulk RNA expression. The flow assays require collection of millions of events but can reliably detect leukemic cells as low as 0.1% to 0.01% (1 in 1000 to 1 in 10,000) of total cells.9Loken M.R. Alonzo T.A. Pardo L. Gerbing R.B. Raimondi S.C. Hirsch B.A. Ho P.A. Franklin J. Cooper T.M. Gamis A.S. Meshinchi S. Residual disease detected by multidimensional flow cytometry signifies high relapse risk in patients with de novo acute myeloid leukemia: a report from Children's Oncology Group.Blood. 2012; 120: 1581-1588Crossref PubMed Scopus (210) Google Scholar, 15Borowitz M.J. Wood B.L. Devidas M. Loh M.L. Raetz E.A. Salzer W.L. Nachman J.B. Carroll A.J. Heerema N.A. Gastier-Foster J.M. Willman C.L. Dai Y. Winick N.J. Hunger S.P. Carroll W.L. Larsen E. Prognostic significance of minimal residual disease in high risk B-ALL: a report from Children's Oncology Group study AALL0232.Blood. 2015; 126: 964-971Crossref PubMed Scopus (227) Google Scholar Flow-based MRD assays, however, are not straightforward.17Chen X. Wood B.L. Monitoring minimal residual disease in acute leukemia: technical challenges and interpretive complexities.Blood Rev. 2017; 31: 63-75Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar They require extensive analytic expertise, and because physiologic and pathologic immunophenotypic parameters may vary across laboratories, numerous control samples must be characterized. Not surprisingly, few laboratories are capable of performing this assay for MRD. To expand the patient population for whom a sensitive, reproducible, PCR-based MRD assay may be clinically useful, Mencia-Trinchant et al6Mencia-Trinchant N. Hu Y. Alas M.A. Ali F. Wouters B.J. Lee S. Ritchie E.K. Desai P. Guzman M.L. Roboz G.J. Hassane D.C. Minimal residual disease monitoring of acute myeloid leukemia by massively multiplex digital PCR in patients with NPM1 mutations.J Mol Diagn. 2017; 19: 537-548Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar used ddPCR to amplify mutant NPM1 cDNA. A number of characteristics of the NPM1 locus and its AML-associated mutations make it a particularly attractive target for MRD assays. NPM1 mutations are among the most common in AML, being found in approximately 30% of all patients.1Papaemmanuil E. Gerstung M. Bullinger L. Gaidzik V.I. Paschka P. Roberts N.D. Potter N.E. Heuser M. Thol F. Bolli N. Gundem G. Van Loo P. Martincorena I. Ganly P. Mudie L. McLaren S. O'Meara S. Raine K. Jones D.R. Teague J.W. Butler A.P. Greaves M.F. Ganser A. Dohner K. Schlenk R.F. Dohner H. Campbell P.J. Genomic classification and prognosis in acute myeloid leukemia.N Engl J Med. 2016; 374: 2209-2221Crossref PubMed Scopus (2261) Google Scholar Moreover, NPM1 mutations are disproportionally found in patients with normal karyotype AML (up to 60%), for whom MRD assessment is critical for treatment decisions and for whom translocation-based MRD analyses are not applicable. In contrast to other frequent mutations in AML, such as internal tandem duplications of FLT3, NPM1 mutations are stable throughout the disease,18Jain P. Kantarjian H. Patel K. Faderl S. Garcia-Manero G. Benjamini O. Borthakur G. Pemmaraju N. Kadia T. Daver N. Nazha A. Luthra R. Pierce S. Cortes J. Ravandi F. Mutated NPM1 in patients with acute myeloid leukemia in remission and relapse.Leuk Lymphoma. 2014; 55: 1337-1344Crossref PubMed Scopus (28) Google Scholar so false-negative results caused by mutation loss rarely occur. Mutation of NPM1 is a marker of leukemic transformation and is not typically present in preleukemic clones. Detection of NPM1 mutations, therefore, reflects leukemic MRD and robustly predicts relapse, whereas detection of common preleukemic mutations (eg, mutations in DNMT3A and IDH1/2) have lower correlation with clinical outcome.7Ivey A. Hills R.K. Simpson M.A. Jovanovic J.V. Gilkes A. Grech A. Patel Y. Bhudia N. Farah H. Mason J. Wall K. Akiki S. Griffiths M. Solomon E. McCaughan F. Linch D.C. Gale R.E. Vyas P. Freeman S.D. Russell N. Burnett A.K. Grimwade D. Assessment of minimal residual disease in standard-risk AML.N Engl J Med. 2016; 374: 422-433Crossref PubMed Scopus (512) Google Scholar Although many mutations of NPM1 have been described, these are mostly tetranucleotide insertions clustered near the 3′ end of the gene.19Falini B. Mecucci C. Tiacci E. Alcalay M. Rosati R. Pasqualucci L. La Starza R. Diverio D. Colombo E. Santucci A. Bigerna B. Pacini R. Pucciarini A. Liso A. Vignetti M. Fazi P. Meani N. Pettirossi V. Saglio G. Mandelli F. Lo-Coco F. Pelicci P.G. Martelli M.F. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1425) Google Scholar PCR primers designed against these insertions can be highly specific for mutant alleles and will theoretically have minimal amplification of unmutated sequence compared with primers against single-nucleotide alterations. Finally, NPM1 is expressed at relatively high levels in blasts,8Gorello P. Cazzaniga G. Alberti F. Dell'Oro M.G. Gottardi E. Specchia G. Roti G. Rosati R. Martelli M.F. Diverio D. Lo Coco F. Biondi A. Saglio G. Mecucci C. Falini B. Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations.Leukemia. 2006; 20: 1103-1108Crossref PubMed Scopus (236) Google Scholar so MRD assays using RNA as starting material have a high analyte/cell ratio, thereby increasing the ability of the assay to detect rare leukemic cells. On the basis of all these characteristics of the NPM1 locus, many groups have used PCR-based NPM1 amplification of cDNA or genomic DNA to track MRD. Indeed, shortly after Falini et al19Falini B. Mecucci C. Tiacci E. Alcalay M. Rosati R. Pasqualucci L. La Starza R. Diverio D. Colombo E. Santucci A. Bigerna B. Pacini R. Pucciarini A. Liso A. Vignetti M. Fazi P. Meani N. Pettirossi V. Saglio G. Mandelli F. Lo-Coco F. Pelicci P.G. Martelli M.F. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1425) Google Scholar characterized NPM1 mutations in AML, this same group developed an NPM1-based real-time quantitative RT-PCR assay for MRD assessment.8Gorello P. Cazzaniga G. Alberti F. Dell'Oro M.G. Gottardi E. Specchia G. Roti G. Rosati R. Martelli M.F. Diverio D. Lo Coco F. Biondi A. Saglio G. Mecucci C. Falini B. Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations.Leukemia. 2006; 20: 1103-1108Crossref PubMed Scopus (236) Google Scholar This latter study clearly found that MRD corresponding to <100 copies of mutant NPM1 per 10,000 copies of ABL1 (ie, 1 in 100) was significantly correlated with favorable outcomes. Multiple additional studies have corroborated these initial findings. Among these, a recent study by Ivey et al7Ivey A. Hills R.K. Simpson M.A. Jovanovic J.V. Gilkes A. Grech A. Patel Y. Bhudia N. Farah H. Mason J. Wall K. Akiki S. Griffiths M. Solomon E. McCaughan F. Linch D.C. Gale R.E. Vyas P. Freeman S.D. Russell N. Burnett A.K. Grimwade D. Assessment of minimal residual disease in standard-risk AML.N Engl J Med. 2016; 374: 422-433Crossref PubMed Scopus (512) Google Scholar deserves particular attention. This study tested a cohort of samples collected prospectively and extensively profiled the genotypes of diagnostic samples by next-generation sequencing. Even with this in-depth genetic assessment, MRD assessment after two cycles of AML chemotherapy retained prognostic power. In fact, multivariate models that include genetic profiling at AML diagnosis and MRD determination by NPM1 RT-PCR indicate that the latter remains an independent variable, whereas diagnostic profiling loses significance. This result is not entirely surprising given the robustness of MRD assessment in previous studies and the marginal contribution of next-generation sequencing for AML prognosis.1Papaemmanuil E. Gerstung M. Bullinger L. Gaidzik V.I. Paschka P. Roberts N.D. Potter N.E. Heuser M. Thol F. Bolli N. Gundem G. Van Loo P. Martincorena I. Ganly P. Mudie L. McLaren S. O'Meara S. Raine K. Jones D.R. Teague J.W. Butler A.P. Greaves M.F. Ganser A. Dohner K. Schlenk R.F. Dohner H. Campbell P.J. Genomic classification and prognosis in acute myeloid leukemia.N Engl J Med. 2016; 374: 2209-2221Crossref PubMed Scopus (2261) Google Scholar The method presented by Mencia-Trinchant et al6Mencia-Trinchant N. Hu Y. Alas M.A. Ali F. Wouters B.J. Lee S. Ritchie E.K. Desai P. Guzman M.L. Roboz G.J. Hassane D.C. Minimal residual disease monitoring of acute myeloid leukemia by massively multiplex digital PCR in patients with NPM1 mutations.J Mol Diagn. 2017; 19: 537-548Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar in this issue attempts to improve the technology used to detect low levels of NPM1 by switching the amplification technique from standard real-time quantitative PCR to ddPCR. In this latter assay, the basic real-time quantitative PCR reagents and protocol for NPM1 mutant amplification and amplification-coupled fluorescence are used; however, the bulk reaction is partitioned into thousands or millions of microdroplets containing (if the experiment is properly constructed) zero or one PCR templates per droplet. Fluorescence of each droplet is determined at the completion of PCR, rather than in real time, with the number of fluorescent droplets corresponding to the amount of mutant NPM1 in the input cDNA. Use of microdroplet partitioning and end point fluorescence detection improves the real-time assay in at least two ways. First, low efficiency off target amplification, which may be a significant confounding variable in real-time PCR, is effectively eliminated in ddPCR, thereby improving the precision of the assay.20Huggett J.F. Cowen S. Foy C.A. Considerations for digital PCR as an accurate molecular diagnostic tool.Clin Chem. 2015; 61: 79-88Crossref PubMed Scopus (297) Google Scholar In a clear demonstration of the precision of ddPCR, Mencia-Trinchant et al6Mencia-Trinchant N. Hu Y. Alas M.A. Ali F. Wouters B.J. Lee S. Ritchie E.K. Desai P. Guzman M.L. Roboz G.J. Hassane D.C. Minimal residual disease monitoring of acute myeloid leukemia by massively multiplex digital PCR in patients with NPM1 mutations.J Mol Diagn. 2017; 19: 537-548Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar found a remarkable coefficient of variance of approximately 5% in multiple experiments. The second improvement to MRD detection afforded by ddPCR involves assay standardization and transcript quantitation. Real-time PCR measures bulk sample fluorescence, which then must be converted to an absolute number of input mutant NPM1 cDNA templates. This is done through establishing a standard curve using known input amounts of a plasmid or synthetic DNA template and extrapolating results obtained with patient samples. Although rather straightforward, this process can become quite unwieldy for NPM1 because there are multiple known mutations, each requiring a unique standard curve. The ddPCR method characterized by Mencia-Trinchant et al6Mencia-Trinchant N. Hu Y. Alas M.A. Ali F. Wouters B.J. Lee S. Ritchie E.K. Desai P. Guzman M.L. Roboz G.J. Hassane D.C. Minimal residual disease monitoring of acute myeloid leukemia by massively multiplex digital PCR in patients with NPM1 mutations.J Mol Diagn. 2017; 19: 537-548Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar overcomes this complexity because the number of input templates correlates in absolute terms with the number of fluorescent droplets. This is particularly true with minimal disease when the chances of incorporating multiple NPM1 mutant templates in a single droplet are infinitesimal. Mencia-Trinchant et al6Mencia-Trinchant N. Hu Y. Alas M.A. Ali F. Wouters B.J. Lee S. Ritchie E.K. Desai P. Guzman M.L. Roboz G.J. Hassane D.C. Minimal residual disease monitoring of acute myeloid leukemia by massively multiplex digital PCR in patients with NPM1 mutations.J Mol Diagn. 2017; 19: 537-548Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar add a third advantage to their assay over common quantitative RT-PCR MRD analysis by creating oligonucleotides with degenerate sequence at known insertion sites. They combined these primers into a single multiplex assay that can detect virtually all known and potential AML-associated NPM1 mutations without sacrificing (and even slightly improving) assay precision. The authors confirm the clinical potential of the assay by testing synthetic templates of rare and potential mutations and showing remarkable concordance between expected and actual results. In testing the pooled primers, the authors also found that multiple distinct NPM1 mutations can be detected simultaneously. Overall, Mencia-Trinchant et al6Mencia-Trinchant N. Hu Y. Alas M.A. Ali F. Wouters B.J. Lee S. Ritchie E.K. Desai P. Guzman M.L. Roboz G.J. Hassane D.C. Minimal residual disease monitoring of acute myeloid leukemia by massively multiplex digital PCR in patients with NPM1 mutations.J Mol Diagn. 2017; 19: 537-548Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar found that the NPM1 mutant ddPCR has sufficient analytic sensitivity and specificity for detection of AML MRD. They present a few patient examples; however, if ddPCR of NPM1 is to be used as a clinical test, further validation is clearly required. The authors argue that precision of the assay, its broad applicability, and relative ease of ddPCR standardization are unique advantages that could allow ddPCR of NPM1 to be widely used in a clinical setting for AML MRD detection. Whether these advantages represent a significant advance in MRD assessment to justify the significant cost of equipment and consumables should be taken into consideration. Real-time PCR of NPM1 performs more than adequately for MRD assessment,7Ivey A. Hills R.K. Simpson M.A. Jovanovic J.V. Gilkes A. Grech A. Patel Y. Bhudia N. Farah H. Mason J. Wall K. Akiki S. Griffiths M. Solomon E. McCaughan F. Linch D.C. Gale R.E. Vyas P. Freeman S.D. Russell N. Burnett A.K. Grimwade D. Assessment of minimal residual disease in standard-risk AML.N Engl J Med. 2016; 374: 422-433Crossref PubMed Scopus (512) Google Scholar and many clinical laboratories already possess the equipment and the technical expertise of real-time analysis. Although the multiplexing of the ddPCR increases the number of patients for whom the assay could be used, most patients possess one of only three mutations,19Falini B. Mecucci C. Tiacci E. Alcalay M. Rosati R. Pasqualucci L. La Starza R. Diverio D. Colombo E. Santucci A. Bigerna B. Pacini R. Pucciarini A. Liso A. Vignetti M. Fazi P. Meani N. Pettirossi V. Saglio G. Mandelli F. Lo-Coco F. Pelicci P.G. Martelli M.F. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1425) Google Scholar so the ability to detect rarer mutations may be marginal from a clinical laboratory (though not an individual patient) perspective. In addition, primer mixes to detect multiple NPM1 mutations have been used in real-time PCR assays.21Barakat F.H. Luthra R. Yin C.C. Barkoh B.A. Hai S. Jamil W. Bhakta Y.I. Chen S. Medeiros L.J. Zuo Z. Detection of nucleophosmin 1 mutations by quantitative real-time polymerase chain reaction versus capillary electrophoresis: a comparative study.Arch Pathol Lab Med. 2011; 135: 994-1000Crossref PubMed Scopus (16) Google Scholar Unfortunately, Mencia-Trinchant et al6Mencia-Trinchant N. Hu Y. Alas M.A. Ali F. Wouters B.J. Lee S. Ritchie E.K. Desai P. Guzman M.L. Roboz G.J. Hassane D.C. Minimal residual disease monitoring of acute myeloid leukemia by massively multiplex digital PCR in patients with NPM1 mutations.J Mol Diagn. 2017; 19: 537-548Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar do not report whether their primer mix can be used in a real-time assay. Perhaps most importantly, centers are increasing the number of tests that they perform on next-generation sequencing platforms, which can have remarkable sensitivity for restricted targets. MRD assessment by next-generation sequencing is currently used clinically for B-lymphoblastic leukemia and is better than multiparametric flow cytometry for outcome prediction.22Pulsipher M.A. Carlson C. Langholz B. Wall D.A. Schultz K.R. Bunin N. Kirsch I. Gastier-Foster J.M. Borowitz M. Desmarais C. Williamson D. Kalos M. Grupp S.A. IgH-V(D)J NGS-MRD measurement pre- and early post-allotransplant defines very low- and very high-risk ALL patients.Blood. 2015; 125: 3501-3508Crossref PubMed Scopus (145) Google Scholar Although Mencia-Trinchant et al6Mencia-Trinchant N. Hu Y. Alas M.A. Ali F. Wouters B.J. Lee S. Ritchie E.K. Desai P. Guzman M.L. Roboz G.J. Hassane D.C. Minimal residual disease monitoring of acute myeloid leukemia by massively multiplex digital PCR in patients with NPM1 mutations.J Mol Diagn. 2017; 19: 537-548Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar argue that results from sequencing may not conform easily to the standard reporting of NPM1 copies per 104 ABL1 copies, this convention is based on history and technology rather than biology, and the convention can surely change to accommodate emerging methods. MRD detection by ddPCR may, therefore, be trapped between two competing technologies that may prevent it from be widely adopted. Real-time PCR performs sufficiently well and is sufficiently entrenched in clinical laboratories to justify a transition to ddPCR. Next-generation digital sequencing with its broad applicability to more than MRD assays and its ever-decreasing marginal costs with increasing testing may obviate the benefits of ddPCR. However, if droplet PCR assays can be established in the regular workflow of a clinical laboratory as stand-alone tests or coupled to downstream sequencing analysis, the NPM1 MRD ddPCR assay would merit further exploration. Calvin Broadus, Chad Hugo, and Pharrell Williams inspired the title but did not contribute to the writing of this manuscript. Minimal Residual Disease Monitoring of Acute Myeloid Leukemia by Massively Multiplex Digital PCR in Patients with NPM1 MutationsThe Journal of Molecular DiagnosticsVol. 19Issue 4PreviewThe presence of minimal residual disease (MRD) is widely recognized as a powerful predictor of therapeutic outcome in acute myeloid leukemia (AML), but methods of measurement and quantification of MRD in AML are not yet standardized in clinical practice. There is an urgent, unmet need for robust and sensitive assays that can be readily adopted as real-time tools for disease monitoring. NPM1 frameshift mutations are an established MRD marker present in half of patients with cytogenetically normal AML. Full-Text PDF Open Archive
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