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From www.bloodjournal.org by guest on June 12, 2017. For personal use only. documented outcome of allogeneic hematopoietic stem cell transplantation, described significantly worse outcomes when haploidentical donors were used compared with genoidentical donors.9 Given that most patients do not have an identical sibling donor available and that enzyme replacement is suboptimal, ADA-deficient SCID was also an ideal model for gene therapy. At the same time that the X-linked SCID trials were being developed, retroviral vector gene therapy was introduced for ADA deficiency. Initial reports were encouraging, but in this issue of Blood, Cicalese and colleagues report on the medium-term survival, immune reconstitution, adverse effects, growth, and development in a cohort of 18 patients who received autologous CD341-enriched cell fractions that contained CD341 cells transduced with a retroviral vector encoding the human ADA complementary DNA sequence, following low-dose busulfan conditioning. With a median follow-up period of 6.9 years (range, 2.3-13.4 years), survival is 100%, results that compare favorably with PEG-ADA enzyme replacement and allogeneic hematopoietic stem cell transplantation (see figure). Most patients demonstrated restoration of T lymphocytes to normal or just below normal ranges, with evidence of thymopoiesis, sustained genemarked circulating T lymphocytes, normal mitogen-induced T-lymphocyte proliferation, B-lymphocytopoiesis (albeit with lower-thannormal circulating B-lymphocyte numbers), and, in some, antibody production to killed and live vaccine antigen and to natural varicella-zoster virus infection. Importantly, although lymphocyte numbers were not completely normalized in all patients, there was evidence of purine metabolite detoxification comparable to that seen in allogeneic hematopoietic stem cell transplantation. The rate of severe infections significantly decreased after gene therapy, and although there was no clear improvement in growth or neurological events, this would not be expected for a hematopoietic stem cell procedure. In 3 patients, the procedure was not considered successful, and although gene transduction was documented, PEG-ADA was recommenced due to poor immune reconstitution; 2 patients have subsequently undergone successful matched sibling donor hematopoietic stem cell transplantation. Critically, although there was evidence of random vector insertion at or near oncogene sites, persisting in the medium-term, 8 no adverse events have been demonstrated at the laboratory or clinical level to date. This study is important for several reasons. Firstly, the participants in the trial included patients who had previously failed haploidentical hematopoietic stem cell transplantation or who had been on PEG-ADA for several months to years and were successfully treated, unlike early gene therapy trials for ADA deficiency. Secondly, the overall results compare extremely favorably to hematopoietic stem cell transplantation, including when using genoidentical sibling donors. Thirdly, despite using similar retroviral vectors to other primary immunodeficiency (PID) gene therapy trials in which mutagenesis was observed, no oncogenic effects have been observed in over 131 follow-up patient-years. The concept of curing inborn errors by use of genetically modified autologous cells is attractive, although longer-term follow-up studies will be required to determine durability of immune reconstitution and immune competence, ongoing safety of gene-transduced cells, and long-term effects of even low-dose alkylating agents such as busulfan. With the issuing of a positive opinion recommending marketing authorization from the Committee for Medicinal Products for Human Use of the European Medicines Agency for an ADA gene therapy vector,10 ADA deficiency may be the first primary immunodeficiency in which gene therapy becomes the “standard of care.” From a disease that, 50 years ago, was universally fatal, and for which there was no available treatment, we are moving to an era where almost 100% survival is expected using gene-corrected autologous cells to provide a lifelong cure. For gene therapy in the field of primary immunodeficiency, this is not the beginning of the end but, truly, the end of the beginning. Conflict-of-interest disclosure: The author declares no competing financial interests. n REFERENCES 1. Cicalese MP, Ferrua F, Castagnaro L, et al. Update on the safety and efficacy of retroviral gene therapy for immunodeficiency due to adenosine deaminase deficiency. Blood. 2016;128(1):45-54. 2. Picard C, Al-Herz W, Bousfiha A, et al. Primary immunodeficiency diseases: an update on the classification from the International Union of Immunological Societies Expert Committee for Primary Immunodeficiency 2015. J Clin Immunol. 2015;35(8):696-726. 3. Gennery AR, Slatter MA, Grandin L, et al. Transplantation of haematopoietic stem cells and long term survival for primary immunodeficiencies in Europe: entering a new century, do we do better? J Allergy Clin Immunol. 2010;126:602-610. 4. Kwan A, Abraham RS, Currier R, et al. Newborn screening for severe combined immunodeficiency in 11 screening programs in the United States. JAMA. 2014;312(7):729-738. 5. Stephan V, Wahn V, Le Deist F, et al. Atypical X-linked severe combined immunodeficiency due to possible spontaneous reversion of the genetic defect in T cells. N Engl J Med. 1996;335(21):1563-1567. 6. Hacein-Bey-Abina S, Garrigue A, Wang GP, et al. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J Clin Invest. 2008;118(9):3132-3142. 7. Hirschhorn R, Martiniuk F, Rosen FS. Adenosine deaminase activity in normal tissues and tissues from a child with severe combined immunodeficiency and adenosine deaminase deficiency. Clin Immunol Immunopathol. 1978;9(3):287-292. 8. Chaffee S, Mary A, Stiehm ER, Girault D, Fischer A, Hershfield MS. IgG antibody response to polyethylene glycolmodified adenosine deaminase in patients with adenosine deaminase deficiency. J Clin Invest. 1992;89(5):1643-1651. 9. Hassan A, Booth C, Brightwell A, et al; Inborn Errors Working Party of the European Group for Blood and Marrow Transplantation and European Society for Immunodeficiency. Outcome of hematopoietic stem cell transplantation for adenosine deaminase-deficient severe combined immunodeficiency. Blood. 2012;120(17):3615-3624, quiz 3626. 10. GlaxoSmithKline. GSK receives positive CHMP opinion in Europe for Strimvelis™, the first gene therapy to treat very rare disease, ADA-SCID. Available at: http://www.gsk.com/ en-gb/media/press-releases/2016/gsk-receives-positivechmp-opinion-in-europe-for-strimvelis-the-first-gene-therapyto-treat-very-rare-disease-ada-scid/. Accessed May 11, 2016. DOI 10.1182/blood-2016-05-715037 © 2016 by The American Society of Hematology l l l MYELOID NEOPLASIA Comment on McKerrell et al, page e1 Just 1 test to diagnose AML?!! ----------------------------------------------------------------------------------------------------Richard Dillon and David Grimwade KING’S COLLEGE LONDON In this issue of Blood, McKerrell et al describe a novel next-generation sequencing (NGS)–based platform for the identification of point mutations, common fusion genes, and copy-number alterations in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) from a single genomic DNA sample.1 BLOOD, 7 JULY 2016 x VOLUME 128, NUMBER 1 From www.bloodjournal.org by guest on June 12, 2017. For personal use only. A different methodologies. It is now clear that AML is driven by a wide range of genomic abnormalities2 and knowledge of these is increasingly being used to personalize lthough the diagnosis of MDS is increasingly shifting to panel-based targeted sequencing, the laboratory workup of AML has traditionally involved a variety of therapy and optimize outcomes for patients. Cytogenetic abnormalities provide powerful prognostic information3 and can identify patients suitable for targeted therapies such Morphology Immunophenotype A B Now The future ? Cells DNA NGS (eg, Karyogene) Karyotype FISH DNA PCR 80 NGS 100 120 140 160 180 200 220 240 10000 5000 0 Fragment analysis Sanger sequencing 1.000 1.000 E-1 1.000 E-2 1.000 E-3 RNA 1.000 E-4 0 10 20 30 40 50 Cycle RT-PCR or RT-qPCR The standard battery of assays required at diagnosis for AML (A) may soon be replaced by a single NGS-based test such as Karyogene (B), described in this issue. FISH, fluorescence in situ hybridization; RT-PCR, reverse transcription PCR. BLOOD, 7 JULY 2016 x VOLUME 128, NUMBER 1 9 From www.bloodjournal.org by guest on June 12, 2017. For personal use only. as all-trans retinoic acid and arsenic trioxide in acute promyelocytic leukemia,4 and gemtuzumab ozogamicin in patients with core-binding factor leukemia.5 Molecular abnormalities provide further prognostic information in those patients lacking a favorable-risk karyotype, also highlighting patients who may benefit from emerging therapies such as smallmolecule inhibitors of Fms-like tyrosine kinase 3 (FLT3) and isocitrate dehydrogenase 1/2 (IDH1/2). In terms of risk stratification, the current European LeukemiaNet guidelines recommend molecular screening for NPM1 mutations and biallelic mutations involving CEBPA, which, in the absence of FLT3–internal tandem duplication (ITD), define further subsets of AML with relatively favorable prognosis.6 Moreover, detection of fusion genes and molecular abnormalities, such as NPM1 exon 12 insertion/deletion (indel) mutations, allows patients to be selected for molecular monitoring using sensitive techniques such as real-time quantitative polymerase chain reaction (qPCR), permitting response-based individualization of treatment.7,8 To deliver optimal therapy and accurately identify patients likely to benefit from allogeneic stem cell transplantation, these genomic abnormalities must therefore be accurately defined at diagnosis; at the present time, this requires a range of different assays to be performed (see figure). Current international guidelines6 stipulate a conventional karyotype, FISH, and molecular testing for a limited number of mutations (NPM1, CEBPA, FLT3-ITD). This has traditionally involved performance of individual DNA- or RNA-based PCR assays and Sanger sequencing. However, many centers now perform NGS panel-based testing for frequently mutated genes to further refine prognosis. This complex array of testing may pose challenges to less specialized laboratories and those with more limited resources. McKerrell and colleagues now present a potential method to simplify diagnostic testing for AML designated “Karyogene”; this system uses sequence capture and NGS coupled with a range of open-source bioinformatic tools to test for common AMLassociated translocations, mutations (in 49 genes recurrently mutated in myeloid 10 malignancy), and copy-number variations and identify regions of copy-neutral loss of heterozygosity from a single sample of genomic DNA. In analysis of a cohort of 112 diagnostic samples (62 AML, 50 MDS), Karyogene could successfully detect the commonest fusion genes including PMLRARA, CBFB-MYH11, RUNX1RUNX1T1, and KMT2A (mixed-lineage leukemia [MLL]) fusions, which together account for ;80% of known AMLassociated translocations; the authors reported 100% sensitivity and specificity for these abnormalities. Importantly, Karyogene was also able to identify 1 rare and 1 novel KMT2A fusion, neither of which were detected by the standard diagnostic workup. Important clinically actionable substitutions and indel mutations in NPM1, FLT3, IDH1, IDH2, DNMT3A, and CEBPA were reliably detected, including some CEBPA mutations missed by standard sequencing. Detection of tandem duplications using short-read sequencing is notoriously challenging; however, McKerrell et al describe 2 new publicly available bioinformatic tools: FLT3 Tandem Finder (F-TAFI; to identify FLT3-ITDs) and MLL Tandem Finder (M-TAFI; to detect MLL partial tandem duplications). When used in conjunction with the Karyogene capture panel, these tools were associated with 100% sensitivity and specificity compared with fragment analysis. Karyogene also performed reasonably well in detecting copy-number changes, showing concordance with the karyotype in 93% of cases. In some cases, small but clinically important deletions (eg, involving 17p [leading to TP53 deletion]) not detected by cytogenetics were found. Although NGS-based platforms such as Karyogene provide an important advance in diagnostic testing for AML, it may be premature for such systems to replace standard cytogenetic testing. Rarer clinically relevant chromosomal rearrangements, such as inv(3)(q21q26)/t(3;3)(q21;q26)/GATA2-EVI1, t(5;11)(q35;p15.5)/NUP98-NSD1, t(6;9)(p23; q34)/DEK-NUP214, t(8;16)(p11;p13)/ MYST3-CREBBP, and t(9;22)(q34;q11)/ BCR-ABL, were not detectable in the current iteration of the panel. However, there would clearly be scope to expand the panel to address this. Another key limitation to implementation of this approach is the level of bioinformatic expertise, which is likely to be beyond that currently available in many diagnostic laboratories. These challenges are undoubtedly surmountable and the results reported by McKerrell and colleagues provide an important proof of principle, offering a glimpse of how hematologic malignancies might be diagnosed in the future. However, with increasing interest in NGS-based diagnostics, it is critical to ensure that RNA (as well as DNA) is routinely extracted at diagnosis to provide baseline samples to allow molecular monitoring of minimal residual disease, which can provide an important tool to further refine outcome prediction.7,8 Conflict-of-interest disclosure: The authors declare no competing financial interests. n REFERENCES 1. McKerrell T, Moreno T, Ponstingl H, et al. Development and validation of a comprehensive genomic diagnostic tool for myeloid malignancies. Blood. 2016;128(1):e1-e9. 2. Grimwade D, Ivey A, Huntly BJ. Molecular landscape of acute myeloid leukemia in younger adults and its clinical relevance. Blood. 2016;127(1):29-41. 3. Grimwade D, Hills RK, Moorman AV, et al; National Cancer Research Institute Adult Leukaemia Working Group. Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood. 2010;116(3): 354-365. 4. Lo-Coco F, Avvisati G, Vignetti M, et al; Gruppo Italiano Malattie Ematologiche dell’Adulto; GermanAustrian Acute Myeloid Leukemia Study Group; Study Alliance Leukemia. Retinoic acid and arsenic trioxide for acute promyelocytic leukemia. N Engl J Med. 2013;369(2): 111-121. 5. Burnett AK, Hills RK, Milligan D, et al. Identification of patients with acute myeloblastic leukemia who benefit from the addition of gemtuzumab ozogamicin: results of the MRC AML15 trial. J Clin Oncol. 2011;29(4):369-377. 6. Döhner H, Estey EH, Amadori S, et al; European LeukemiaNet. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood. 2010;115(3): 453-474. 7. Ivey A, Hills RK, Simpson MA, et al; UK National Cancer Research Institute AML Working Group. Assessment of minimal residual disease in standard-risk AML. N Engl J Med. 2016;374(5): 422-433. 8. Grimwade D, Freeman SD. Defining minimal residual disease in acute myeloid leukemia: which platforms are ready for “prime time”? Blood. 2014; 124(23):3345-3355. DOI 10.1182/blood-2016-05-715060 © 2016 by The American Society of Hematology BLOOD, 7 JULY 2016 x VOLUME 128, NUMBER 1 From www.bloodjournal.org by guest on June 12, 2017. For personal use only. 2016 128: 8-10 doi:10.1182/blood-2016-05-715060 Just 1 test to diagnose AML?!! 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