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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
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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?!!
Richard Dillon and David Grimwade
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