The n e w e ng l a n d j o u r na l of m e dic i n e
Brief Report
From Bluebird Bio, Cambridge, MA (S.G.,
D.W., H.B., P.D.G., G.P., M.F., A.Y., M.G.,
S.B.V., A.M., J.L., R.A.C., M.B.); the Cel-
lular and Molecular Therapeutics Branch,
National Heart, Lung, and Blood Insti-
tute–National Institute of Diabetes and
Digestive and Kidney Diseases, National
Institutes of Health, Bethesda, MD (J.T.);
GeneWerk, Heidelberg, Germany (M.S.);
the University of Alabama at Birming-
ham, Birmingham (J.K.); and the Division
of Pediatric Hematology–Oncology, Med-
ical University of South Carolina, Charles-
ton (J.J.). Dr. Bonner can be contacted at
­mbonner@​­bluebirdbio​.­com or at Bluebird
Bio, 60 Binney St., Cambridge, MA 02142.
This article was published on December
12, 2021, at NEJM.org.
N Engl J Med 2022;386:138-47.
DOI: 10.1056/NEJMoa2109167
Copyright © 2021 Massachusetts Medical Society.
138
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Acute Myeloid Leukemia Case after Gene
Therapy for Sickle Cell Disease
Sunita Goyal, M.D., John Tisdale, M.D., Manfred Schmidt, Ph.D.,
Julie Kanter, M.D., Jennifer Jaroscak, M.D., Dustin Whitney, Ph.D.,
Hans Bitter, Ph.D., Philip D. Gregory, Ph.D., Geoffrey Parsons, Ph.D.,
Marianna Foos, M.S., Ashish Yeri, Ph.D., Maple Gioia, A.L.M.,
Sarah B. Voytek, Ph.D., Alex Miller, B.S., Jessie Lynch, M.S.,
Richard A. Colvin, M.D., Ph.D., and Melissa Bonner, Ph.D.​​
Sum m a r y
Gene therapy with LentiGlobin for sickle cell disease (bb1111, lovotibeglogene
autotemcel) consists of autologous transplantation of a patient’s hematopoietic stem
cells transduced with the BB305 lentiviral vector that encodes the βA-T87Q-globin
gene. Acute myeloid leukemia developed in a woman approximately 5.5 years after
she had received LentiGlobin for sickle cell disease as part of the initial cohort
(Group A) of the HGB-206 study. An analysis of peripheral-blood samples revealed
that blast cells contained a BB305 lentiviral vector insertion site. The results of an
investigation of causality indicated that the leukemia was unlikely to be related to
vector insertion, given the location of the insertion site, the very low transgene
expression in blast cells, and the lack of an effect on expression of surrounding
genes. Several somatic mutations predisposing to acute myeloid leukemia were
present after diagnosis, which suggests that patients with sickle cell disease are at
increased risk for hematologic malignant conditions after transplantation, most
likely because of a combination of risks associated with underlying sickle cell disease,
transplantation procedure, and inadequate disease control after treatment. (Funded
by Bluebird Bio.)
S
ickle cell disease is a progressive, genetic disease caused by a
point mutation in the β-globin gene and is associated with substantial mor-
bidity and early mortality.1,2 Sickle hemoglobin production and subsequent
polymerization leads to red-cell sickling, clinically manifesting as chronic hemo-
lytic anemia, vasculopathy, and vaso-occlusion.1,2 Current management of sickle
cell disease relies on lifelong use of palliative short-term and long-term thera-
pies.1,2 Allogeneic hematopoietic stem-cell transplantation is the only accepted,
potentially curative treatment option for patients with sickle cell disease; however,
its use is limited by a paucity of matched donors, immunologic complications, and
the inherent risks of transplantation-related morbidity and mortality.1,3,4
Gene therapy with LentiGlobin for sickle cell disease (bb1111, lovotibeglogene
autotemcel) consists of autologous transplantation of the patients’ own hemato-
poietic stem cells (HSCs) transduced with the BB305 lentiviral vector encoding the
βA-T87Q-globin gene, which is designed to produce antisickling hemoglobin (HbAT87Q).5-7
Through the use of autologous HSCs, gene therapy with LentiGlobin circumvents
the need for a donor and may avoid the immunologic complications associated with
allogeneic HSC transplantation. The phase 1–2 HGB-206 study is evaluating the ef-
ficacy and safety of LentiGlobin for sickle cell disease, and an unprespecified in-
n engl j med 386;2 nejm.org January 13, 2022
The New England Journal of Medicine
 Brief Report
terim analysis of the results is now reported in
the Journal.8 Here, we report a case of acute my-
eloid leukemia that developed in a female patient
approximately 5.5 years after she had received
gene therapy with LentiGlobin as part of the ini-
tial cohort (Group A) of the HGB-206 study, as
well as the results of investigations to deter-
mine whether the development of acute myeloid
leukemia was vector-mediated.
C a se R ep or t
Before receiving gene therapy with LentiGlobin,
the patient had sickle cell disease (βS/βS geno-
type) with recurrent vaso-occlusive crises that had
led to frequent hospitalizations despite the use of
long-term hydroxyurea therapy. She was 31 years
of age when she received a diagnosis of acute
myeloid leukemia in February 2021 (Table 1).
In August 2015, as part of the Group A cohort
in the HGB-206 study, the patient received Lenti­
Globin produced from HSCs harvested from bone
marrow through an earlier version of the drug-
product manufacturing process as part of the
initial study protocol. The manufacturing process
was later modified for subsequent patients en-
rolled in Groups B and C (Table S1 in the Sup-
plementary Appendix, available with the full text
of this article at NEJM.org). The drug product
administered in the patient had a total CD34+
cell dose of 2.6 × 106 cells per kilogram of body
weight (of which 1.5 × 106 cells per kilogram were
CD34hi long-term repopulating hematopoietic stem
and progenitor cells [HSPCs]) and a vector copy
number of 0.57 copies per diploid genome (Ta-
ble 1). At day 16 after LentiGlobin treatment, the
patient received granulocyte colony-stimulating
factor because of fever, and subsequent neutro-
phil and platelet engraftments occurred at 19
and 31 days after treatment, respectively. At 6
months after treatment, the vector copy number
in peripheral blood was 0.05 copies per diploid
genome and the level of gene therapy–derived
hemoglobin (HbAT87Q) was 4.33% of total non-
transfused hemoglobin, findings that suggest low
engraftment of gene-modified cells and low trans-
gene expression with minimal clinical benefit,
because the pretransplantation sickle cell disease
phenotype persisted and led to the need for
hydroxyurea therapy and packed red-cell trans-
fusions (Table 1 and Table S2).
During an emergency department visit for a
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vaso-occlusive crisis in December 2020, the patient
was noted to have 2% blast cells in peripheral
blood, which was thought to be related to recov-
ery after infection; the presence of blast cells
was not reported in subsequent blood analyses.
In January 2021, neutropenia (defined as an ab-
solute neutrophil count of <1000 per microliter)
developed, and in February 2021, acute myeloid
leukemia (subtype M0 according to the French–
American–British classification) was diagnosed
after the detection of 29% blast cells in peripheral
blood and 22 to 50% blast cells in bone marrow
(Table 1). Given the potential of insertional on-
cogenesis by the lentiviral vector and the expo-
sure to myelotoxic chemotherapy with busulfan,
the acute myeloid leukemia was studied to deter-
mine causality and relation to gene therapy. Chro-
mosomal microarray analysis of the bone marrow
samples revealed monosomy 7 in 70% of cells
and partial loss of 11p involving WT1 in 50% of
cells. A next-generation sequencing myeloid mu-
tation panel revealed a RUNX1 frameshift muta-
tion and a PTPN11 missense mutation (Table 1).
Retrospective next-generation sequencing and
single-nucleotide-polymorphism microarray anal-
yses of retained pretreatment peripheral-blood
leukocytes did not reveal any genetic abnormali-
ties, nor were any abnormalities observed in any
retained post-treatment sample up to month 48
(the most recent sample obtained before the di-
agnosis of acute myeloid leukemia). Further anal-
ysis of the peripheral-blood samples revealed that
the blast cells were positive for BB305 lentiviral
vector. Specifically, CD34+ blast-enriched cell pop-
ulations in samples of both peripheral-blood and
bone marrow contained an integrated lentiviral
vector, with vector copy numbers of 1.12 copies
per diploid genome and 1.17 copies per diploid
genome, respectively. The percentage of lentivi-
ral vector–positive cells was 92.1% in the CD34+
peripheral-blood sample and 86.7% in the CD34+
bone marrow sample. The investigator assessed
the acute myeloid leukemia as possibly related to
her gene therapy.
After acute myeloid leukemia was diagnosed,
the patient underwent three cycles of induction
chemotherapy, which led to morphologic remis-
sion, but she remained positive for minimal resid-
ual disease with a blast-cell percentage of 0.2%.
She underwent a human leukocyte antigen–hap-
loidentical peripheral-blood stem-cell transplan-
tation in June 2021 and remained positive for
nejm.org January 13, 2022 139
 The n e w e ng l a n d j o u r na l of m e dic i n e

Table 1. Baseline and Treatment Characteristics and Clinical Outcomes in a Patient with AML after LentiGlobin Treatment for Sickle Cell Disease.*

Variable Patient Information

Demographic characteristic
Age at treatment (date of treatment) 25 yr (Aug. 2015)
Sex Female
Genotype βS/βS
Race Black
Family history of AML or myelodysplastic syndrome No
Smoking history Never smoked
Clinical feature of sickle cell disease in the 2 yr preceding
consent
Hydroxyurea use Yes (approximately 6 yr of long-term use; treatment was discontinued
approximately 6 mo before LentiGlobin treatment)
Regular transfusions of packed red cells No
Stroke No
Previous complications of sickle cell disease Recurrent vaso-occlusive crisis, osteonecrosis, avascular necrosis of right
hip, chronic pain due to sickle cell disease, deep-vein thrombosis
Transplantation characteristic
Mean estimated area under the plasma 4084 μmol × min
busulfan concentration–time curve
Neutrophil engraftment† 19 days after treatment
Platelet engraftment‡ 31 days after treatment
Drug-product characteristic
Source of hematopoietic stem cells in Group A Manufactured from bone marrow harvest§
Mean VCN in the drug product 0.57 copies/diploid genome
Total CD34+ cell dose 2.6 × 106/kg
Long-term hematopoietic stem and progenitor cells (CD34 ) hi
1.5 × 106/kg
Post-treatment clinical course
VCN in peripheral blood at 6 mo 0.05 copies/diploid genome
Gene therapy–derived hemoglobin (HbAT87Q) level at 6 mo 4.33% of nontransfused total hemoglobin
Clinical signs and complications No improvement in hemolysis, continued vaso-occlusive crisis, chronic pain
Sickle cell disease–related concomitant medications and Granulocyte colony-stimulating factor (days 16 and 17 after treatment),
transfusions regular transfusions of packed red cells, hydroxyurea (days 186–229,
282–520, and 1018–1754 after treatment)
AML diagnosis
Interval between treatment and diagnosis (date of diagnosis) Approximately 5.5 yr (Feb. 2021)
Clinical course leading to AML diagnosis
Dec. 28, 2020 2% Blast cells in peripheral blood (resolved, thought to be due to infection
recovery); white-cell count, 7030 per microliter
Feb. 2, 2021 9% Blast cells in peripheral blood; white-cell count, 6450 per microliter
Feb. 8, 2021 29% Blast cells in peripheral blood; white-cell count, 10,900 per microliter
Feb. 9, 2021 22–50% Blast cells in bone marrow with confirmed diagnosis of AML (sub-
type M0 according to the French–American–British classification); white-
cell count, 12,800 per microliter
Feb. 9, 2021 Analysis of bone marrow identified RUNX1 exon 5 stop-gained p.A149*fs
(VAF, 26%); PTPN11 exon 3 missense p.A72V (VAF, 30%); monosomy 7
(70% of cells); partial loss of 11p (50% of cells)

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 Brief Report

Table 1. (Continued.)

Variable Patient Information

Vector and blast-cell analysis
CD34+ blast cells determined to be lentiviral vector–positive
VCN in peripheral blood 1.12 copies/diploid genome (92.1% LVV+ cells)
VCN in bone marrow 1.17 copies/diploid genome (86.7% LVV+ cells)
Integration-site analysis Vector in blast cells with a single insertion site in VAMP4

* AML denotes acute myeloid leukemia, LVV+ lentiviral vector–positive, VAF variant allele frequency, and VCN vector copy number.
† Neutrophil engraftment was defined as the first day of absolute neutrophil count of at least 500 per microliter for three consecutive mea-
surements on different days after the initial post-transplant nadir by day 43, without receiving backup cells at any time during the neutrope-
nic phase.
‡ Platelet engraftment was defined as the first of 3 consecutive platelet counts of at least 50,000 per microliter on different days after the ini-
tial post-treatment nadir, without platelet transfusions for 7 days immediately preceding and during the evaluation period.
§ The drug-product manufacturing process was later modified for subsequent patients enrolled in Groups B and C in the HGB-206 study.

minimal residual disease. The patient had a re-
lapse with the reappearance of circulating blast
cells 90 days after transplantation and underwent
additional chemotherapy; however, she later died
from complications of progressive acute myeloid
leukemia.
Me thods
The phase 1–2 HGB-206 study evaluated the use
of LentiGlobin in patients with sickle cell disease.
The drug product was manufactured by trans-
duction of autologous enriched CD34+ stem
cells with BB305 lentiviral vector encoding the
antisickling βA-T87Q-globin gene. The structure of
the BB305 lentiviral vector has been previously
described.5 To assess whether the acute myeloid
leukemia in the patient was due to insertional
oncogenesis, we performed integration site analy-
sis and RNA sequencing, as described previous-
ly9-13 and detailed in the Supplementary Appendix.
R e sult s
Integration Site Analysis
We performed an integration site analysis to
evaluate the role of vector insertion in the devel-
opment of acute myeloid leukemia. Many unique
insertion sites (a total of 2997) were detected in
the patient; however, the insertion site in the
vesicle-associated membrane protein 4 gene
(VAMP4) increased over time, reaching a relative
frequency of more than 60% in August 2020
(Fig. 1A) and 98.6% in February 2021. The in-
crease in VAMP4 insertion-site frequency prompt-
ed specific quantitative polymerase-chain-reac-
tion analyses designed to estimate the fractional
contribution of the clone carrying the VAMP4
insertion site in peripheral blood. Until January
2020 (54 months after treatment), the VAMP4
insertion site was present in approximately 1%
of peripheral-blood cells in the patient, and by
August 2020, this increased to 7% of peripheral-
blood cells (Fig. 1B). This increase coincided
with an overall increase in the vector copy num-
ber in peripheral blood during this period,
which increased from approximately 0.05 copies
per diploid genome to 0.13 copies per diploid
genome. Concurrent with this rise, decreases in
white-cell and absolute neutrophil counts were
also observed; these findings suggest a potential
correlation of these measures (Fig. 1C).
A single insertion was identified in VAMP4 in
the intronic region between exons 4 and 5 that
did not disrupt a putative enhancer (approxi-
mately 5 kilobases upstream) or the promoter
(approximately 30 kilobases upstream). An in-
sertion site in the VAMP4 gene body was present
in most patients (25 of 35 [71%]) in the HGB-206
study who had received LentiGlobin and had
available data from integration site analysis. A
total of 60 unique insertions in the VAMP4 gene
locus were mapped, of which 14 were localized
to the intronic region between exons 4 and 5,
including the VAMP4 insertion site in the patient
with acute myeloid leukemia (Fig. 1D). As com-
pared with other protein-coding genes, VAMP4
was in the 68th percentile for insertion sites when
normalized for gene length. In all other patients
in the HGB-206 study, VAMP4 insertion sites were

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 The n e w e ng l a n d j o u r na l of m e dic i n e

A B
100 VAMP4 1.0

PB VCN (copies/diploid genome)

90 MSH5
80 RHOT1
Sequence Count (%) 70 LOC100294145
60 WNK1
STOM
50 0.5
FBXW7
40 POLR2G
30 Insertion
NOSIP site–specific
20 VAV1 VCN
10 All other insertion sites Overall VCN

0 0.0
August 2020 0 12 24 36 48 60 72
Months since LentiGlobin Treatment

C
20,000

White-cell count
Cell Count (per microliter)

15,000

10,000

5,000
Absolute neutrophil count

0
0.0 0.5 1.0 12 24 36 48 60 64 65 66 67
Months since LentiGlobin Treatment

D
BB305 VAMP4 insertion sites
in all patients with insertion sites
outside the intronic region between
exons 4 and 5
BB305 VAMP4 insertion sites BB305 VAMP4 insertion site
in all patients with insertion sites in patient with AML
inside the intronic region between
exons 4 and 5

BB305
Insertion Site

VAMP4
Exon 8 Exon 7 Exon 5 Exon 4 Exon 3 Exon 2 Exon 1
Exon 6
17,167× 104 17,168× 104 17,169× 104 17,170× 104 17,171× 104 17,172× 104 17,173× 104
Location on Chromosome 1 (no. of base pairs)

not associated with sequelae, including clonal was similar to that in mobilized peripheral-blood
expansion. CD34+ cells from a healthy donor (Fig. 2A and
Table S3). Next, we measured HBB expression lev-
Gene Expression Analysis els, including lentiviral vector–derived βA-T87Q and
We performed RNA-sequencing analysis to de- endogenous βS transcripts, in different cell frac-
termine the transcriptional effect of the VAMP4 tions. The highest expression levels of HBA1 and
insertion site. Given the location of the insertion HBB were in unsorted and CD34− bone marrow
site, we first determined whether VAMP4 expres- cells, which is expected in cell populations en-
sion had changed. An analysis of the CD34+ cells riched for the presence of erythroblasts and
from the patient showed VAMP4 expression that erythroid progenitors. In contrast, in CD34+ pe-

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 Brief Report

indicated very low expression levels in blast cells.

Figure 1 (facing page). Lentiviral Vector Integration-Site
Among the CD34+ peripheral-blood HBB tran-
Analysis and Cell Counts in a Patient with AML after
LentiGlobin Treatment for Sickle Cell Disease. scripts, the majority (60%) were endogenous βS
transcripts — 40% derived from the therapeutic
Acute myeloid leukemia (AML) developed in a patient
who had been treated in the initial cohort (Group A) of
transgene βA-T87Q. In a finding consistent with
the HGB-206 study of LentiGlobin for sickle cell disease
the site and orientation of vector insertion, we
(bb1111, lovotibeglogene autotemcel).8 Panel A shows
also detected very low levels of HBB–VAMP4 and
the distribution of gene-marked cell clones at the un-
VAMP4–HBB fusion transcripts (<1 normalized
scheduled visit by the patient in August 2020, as deter-
fusion fragment transcript per million for CD34+
mined by integration-site analysis at GeneWerk. Each
of the top 10 most frequently detected unique insertion
and CD34− peripheral-blood samples). These re-
sites are indicated by a different color, with red show-
sults showed that the BB305 lentiviral vector had
ing VAMP4 (64.9%) and gray showing the cumulative
minimal transcriptional activity in blast cells
proportion of all other unique insertion sites (723 sites
and no noticeable effect on VAMP4 expression.
[27.6%]). Other than VAMP4, none of the insertion sites
We investigated whether the VAMP4 insertion
showed a relative frequency of more than 5% of the to-
tal retrieved insertion sites. Panel B shows the overall
site affected gene expression across the 10-mega-
vector copy number (VCN) in peripheral blood (PB),
base region flanking VAMP4. An analysis of the
as determined by central laboratory analysis, and the
CD34+ cells from the patient, as compared with
VAMP4 insertion site–specific PB VCN, as determined
CD34+ cells from a healthy donor, showed gene-
by analysis at GeneWerk. Insertion site–specific VCN
expression ratios centered around 1 (Fig. 2B).
can be used to estimate the fractional contribution of
the VAMP4-containing clone within the analyzed popu-
These population-level data indicated that ex-
lation. Samples were analyzed from scheduled visits
pression levels of genes proximal to the VAMP4
(≤60 months after LentiGlobin treatment) and unsched-
locus in CD34+ bone marrow cells from the
uled visits (>60 months after treatment, August 2020–
patient were similar to those in the CD34+ mo-
February 2021). Panel C shows the white-cell count and
bilized peripheral-blood cells from the healthy
absolute neutrophil count, as determined by central-
laboratory analysis of data from scheduled visits; these
donor. An assessment of genes within 100 kilo-
data are available from the clinical databases of the
bases of the VAMP4 locus revealed no or minimal
HGB-206 study and its long-term follow-up study LTF-
changes in expression level in analyzed genes in
307 (ClinicalTrials.org number, NCT04628585). Analysis
all patient samples, particularly in genes with a
of samples from unscheduled visits (>60 months; De-
transcripts-per-million value of 1.0 or greater
cember 2020–February 2021) were determined by local
laboratory analysis; these data were available from the
(Fig. 2B and Table S3). An analysis of all chro-
safety database of the LTF-307 study. Panel D shows
mosomes showed a ratio of protein-coding gene
the frequency and location of VAMP4 insertions in the
transcripts centered around 1, indicating that
patients treated with LentiGlobin in the HGB-206 study;
the only chromosome-wide differences in gene
integration-site analysis data were derived from an ex-
expression between the patient and the healthy
ploratory research database (first integration-site analy-
sis data at 6 months after treatment). Open black trian-
donor occurred in chromosome 7 (Fig. S2). The
gles represent unique insertion sites, and open red
shift in the gene-expression ratio for chromo-
triangles represent insertion sites inside the intronic
some 7 is consistent with monosomy 7. Finally,
­region between exons 4 and 5, including the VAMP4
targeted whole-genome sequencing analysis of
­insertion site of interest in the patient with AML repre-
the 10-megabase flanking region of the VAMP4
sented by a solid red triangle. The triangles are offset
from each other to enhance visibility. Below the inser-
insertion site showed no chromosomal abnor-
tion sites, the 8 exons of VAMP4 are represented by
malities or copy-number alterations, which sug-
purple bars, and the intronic regions are in gray. VAMP4
gests that BB305 lentiviral vector insertion did not
exon boundaries, derived from the principal isoform
result in unexpected chromosomal damage in this
from the APPRIS (annotating principal splice isoforms)
region (data not shown). These results indicated
database, are extracted from GENCODE, version 36,
and overlaid onto the GRCh37 (hg19) human reference
that gene expression within 10 megabases of the
genome.
VAMP4 insertion site was not noticeably affected,
and no chromosomal abnormalities were ob-
served. The overall transcriptional profile in the
ripheral-blood cells enriched for leukemic blast blast cells was consistent with monosomy 7, a
cells, HBB expression was 0.6% of that detected common genetic lesion in alkylating agent-induced
in the sample of CD34− bone marrow cells, which acute myeloid leukemia.15

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 144
A
VAMP4 HBA1 HBB
Blast- Blast- Blast- Blast- Blast- Blast-
enriched depleted enriched depleted enriched depleted
80 25,000 120,000
60 20,000 90,000
15,000
40 60,000
10,000
20 5,000 30,000
0 0 0
M B P B PB 1 2 PB M B M PB PB 1 2 PB PB B PB 1 2 PB

Transcripts per Million

BM ,B ,P BM −, m ,# ,# BM ,B ,P ,B −, m ,# ,# m BM , BM BM − , P m ,# ,# m
d + + −, , M M ,m d + + − , M M , d + 4 +, −, 4 , M M ,
rte 34 34 34 34 3+ B B + rte 34 34 34 34 3+ ,B ,B 4+ tr e 3 4 3 3 4 3 + B B +
so CD CD CD 3 −, −, 34 so CD CD CD 3 − − 3 so CD CD CD 33 −, −, 34
CD CD 34 34 CD CD CD 34 34 CD CD CD 34 34 CD
Un D D Un Un D D
C C CD CD C C

Patient with AML Healthy Donors Patient with AML Healthy Donors Patient with AML Healthy Donors

B
Chromosome 1 10-Megabase flanking VAMP4
400 25
The

300 20
15
200
10
100

No. of Genes
5
0 0
0 2 4 0 2 4

n engl j med 386;2

Expression Ratio Expression Ratio
DNM3 METTL13 MYOC
Blast- Blast- Blast- Blast- Blast- Blast-

nejm.org
enriched depleted enriched depleted enriched depleted
8 30 25
6 25 20
n e w e ng l a n d j o u r na l

20 15

The New England Journal of Medicine

4 15
of

10 10
2 5 5
0 0 0
B B PB 1 2 PB
BM , BM ,P ,# ,#

January 13, 2022

BM − , P
PFN1P1 RPL4P3 d + 4+ 4 −, 4 M ,m
rte 3 4 3 3 3 +, m BM B +
Blast- Blast- Blast- Blast- so CD CD CD 33 −, −, 34

Copyright © 2022 Massachusetts Medical Society. All rights reserved.

CD CD 34 34 CD
m e dic i n e

enriched depleted enriched depleted Un

0.8 0.4 CD CD
0.6 0.3

Transcripts per Million

Patient with AML Healthy Donors
0.4 0.2
0.2 0.1
0.0 0.0
M PB P B PB 1 2 PB M PB M PB PB 1 2 PB
BM ,B BM −, m,# ,# BM ,B ,B −, m ,# ,# m
d + +, −, , M M ,m d + +, − , M M ,
rte 34 34 34 34 3+ B B + rte 34 34 34 34 3+ ,B ,B 4+
so CD CD CD 3 −, −, 34 so CD CD CD 3 − − 3
CD CD 34 34 CD CD CD 34 34 CD
Un Un
CD CD CD CD

Patient with AML Healthy Donors Patient with AML Healthy Donors

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 Brief Report
Figure 2 (facing page). Gene Expression Analysis
in AML Blast Cells and within 10 Megabases
of the VAMP4 Locus.
Panel A shows the normalized expression levels for
VAMP4, HBA1, and HBB, as determined by bulk RNA-
sequencing analysis. The increased expression of VAMP4
in blast-depleted CD34− bone marrow (BM) cells is
probably driven by CD34+ myeloblasts differentiating
into CD34− erythroid progenitor cells.14 This increased
expression of VAMP4 is supported by the concurrent
increase in HBA1 and HBB expression in this erythro-
blast-containing population, the increase in antisickling
hemoglobin HbAT87Q that coincides with the emergence
of this clone (Fig. S1), and the increased detection of
fusion transcripts between VAMP4 and the transgenic
βA-T87Q in this CD34− BM cell population, the number
of which was more than 10 times as high as that in
CD34− PB cells. The y axis scale was adjusted for each
gene. Panel B shows histograms of gene-expression
ratios on chromosome 1 (upper left) and within 10
megabases of VAMP4 (upper right) comparing CD34+
BM cells from the patient with AML with CD34+ mobi-
lized PB (mPB) cells from a healthy donor. The labels
of the upper-left and upper-right histograms indicate
the compared loci; the x axes represent the expression
ratio between CD34+ BM cells from the patient with
AML (numerator) and CD34+ BM cells from a healthy
donor (denominator); and the y axes denote the num-
ber of genes that populate each binned range. The red
dotted lines indicate an expression ratio of one. The
chromosome for which the distribution peak is on the
dotted line indicates no overall change in protein-coding
gene expression for the region of interest. The bottom
set of graphs shows the normalized expression levels
for the five genes closest to VAMP4 (DNM3, METTL13,
MYOC, PFN1P1, and RPL4P3), as determined by bulk
RNA-sequencing analysis; the y axis scales were ad-
justed for each gene. In Panels A and B, the purity of
sorted fractions ranged from 90.69 to 97.90% (CD34+
PB, 90.69%; CD34− PB, 95.57%; CD34+ BM, 95.38%;
and CD34− BM, 97.90%). CD34+ and CD33+ mPB cells
were derived from a single healthy donor, and each
CD34– BM sample (#1 and #2) was derived from a
different healthy donor.
Discussion
Among the seven patients who received Lenti-
Globin treatment in the initial cohort (Group A)
of the HGB-206 study, this patient is the second
to present with acute myeloid leukemia. Under-
standing potential risk factors for the develop-
ment of hematologic malignant conditions is
important for communities affected by sickle cell
disease and persons who are considering gene
therapy. In contrast to the case of acute myeloid
leukemia that was diagnosed in 2018,15 the case
in the current patient showed vector present in
n engl j med 386;2
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leukemic blast cells, which suggests that blast cells
originated from a transduced HSC and not from
residual unablated host cells exposed to busulfan.
This report provides several lines of evidence
showing that the development of this case of
acute myeloid leukemia most likely occurred in-
dependently of insertional oncogenesis. First,
the insertion site was in VAMP4, a gene that is
known to play a role in Golgi structure and
function16,17 but has no known role in cellular
proliferation or oncogenesis on the basis of an
analysis of the published literature and gene
databases (COSMIC [Catalogue of Somatic Muta-
tions in Cancer], cBioPortal, DepMap [Dependency
Map], and CCGD [Candidate Cancer Gene Data-
base]), which included sequenced cancer tissue
samples from more than 2000 patients with acute
myeloid leukemia and 800 human-derived can-
cer cell lines (see the Supplementary Appendix).
Second, the insertion site mapped to a noncoding
region of VAMP4 in a location with no annotated
genomic features, a finding that is in line with
the frequent integration of lentiviral vector into
introns18 because of their larger size relative to
exons in a gene. Third, a VAMP4 insertion site was
present in most evaluated patients with sickle
cell disease (71%) who received treatment in the
HGB-206 study and had no sequelae. Our findings
are in keeping with those reported for other
lentiviral vectors, which frequently integrate into
VAMP4.19 Finally, the BB305 lentiviral vector was
minimally transcriptionally active in blast cells and
did not noticeably alter the expression of proxi-
mal genes, and no chromosome-wide changes in
gene expression were observed, except in chromo-
some 7, which is consistent with monosomy 7.
Because the development of acute myeloid
leukemia was most likely independent of vector
insertion, other contributing factors in both pa-
tients should be considered. Genetic testing iden-
tified several known driver alterations of acute
myeloid leukemia in the blast cells from the cur-
rent patient (RUNX1, PTPN11, monosomy 7, and
partial loss of 11p), which suggests a molecular
basis for the findings observed in this case. In
the previous case diagnosed in 2018, a similar
mutation profile (RUNX1, PTPN11, and monoso-
my 7) was observed.15 Both patients could have
accumulated several mutations predisposing them
to the development of acute myeloid leukemia
over the course of their sickle cell disease and
their treatment, resulting in a clone that led to
nejm.org January 13, 2022 145
 The n e w e ng l a n d j o u r na l
acute myeloid leukemia. The mechanism behind
the accumulation of these somatic mutations is
likely to be multifactorial, with contributions from
the transplantation procedure and its associated
risks, the underlying sickle cell disease, and the
continued hematopoietic stress resulting from
the minimal clinical benefit derived from Lenti-
Globin, which was observed in both cases.15
Among patients with sickle cell disease, the
risk of hematologic malignant conditions is 2 to
11 times as high as that in the general popula-
tion.20,21 Factors that probably contribute to this
increased baseline risk include the underlying
pathophysiological mechanism of chronic hypoxia,
the generation of reactive oxygen species with as-
sociated oxidative stress, the chronic inflamma-
tion, the endothelial and vascular damage and
constant erythropoietic stress, the high cell turn-
over, and the damage to the HSC compartment
in bone marrow.1,2,20,21 Use of hydroxyurea for
sickle cell disease has been suggested as a pos-
sible risk factor, but analyses of data accumu-
lated over decades from patients with sickle cell
disease and chronic myeloid leukemia have not
established a link.22 The transplantation proce-
dure, including the use of myeloablative condi-
tioning agents such as busulfan, carries inherent
risks of malignant disease.23-25 Furthermore, af-
ter myeloablation, the bone marrow niche under-
goes extensive proliferation of HSCs and HSPCs,
generating proliferative stress that may lead to
the rise and accumulation of mutations as part
of the normal engraftment process.26 If a lower
cell dose is administered to a patient after mye-
loablation, fewer HSCs and HSPCs are available
to repopulate the bone marrow, which leads to
the need for more replication cycles and hence
additional proliferative stress and a higher prob-
ability of acquiring a mutation that could lead to
acute myeloid leukemia.
The two patients in Group A of the HGB-206
study in whom acute myeloid leukemia developed
had received LentiGlobin produced from bone
marrow–harvested HSCs through an earlier ver-
sion of the drug-product manufacturing process.
Cell collections from the bone marrow with the
use of this process resulted in low cell doses,
with fewer CD34hi long-term HSPCs, leading to
increased proliferative stress (Table S1). The low
transduction efficiency of the drug product in
these patients resulted in minimal transgene
146 n engl j med 386;2
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Copyright © 2022 Massachusetts Medical Society. All rights reserved.
of m e dic i n e
expression and an inadequate therapeutic response,
with ongoing hemolysis and persistent anemia
— outcomes that subjected transplanted and
endogenous bone marrow cells to continued he-
matopoietic stress after treatment, providing fur-
ther opportunity for accumulation of mutations.
Before the development of malignant disease
in these two patients, alterations to the HGB-206
study protocol were necessary to improve clinical
benefit; thus, the initial protocol and manufac-
turing process used in Group A underwent sev-
eral changes to improve HSC collection and trans-
duction efficiency. The protocol used in Group C
includes a minimum of 60 days of packed red-cell
transfusions before HSC collection, HSC collec-
tion by mobilization with plerixafor followed by
apheresis, and an increased target value for the
area under the plasma busulfan concentration–
time curve during conditioning. Some of these
modifications have positively affected the quality
(CD34hi) and number of HSCs available for ex vivo
manipulation (Table S1). In addition, the improve-
ment in transduction efficiency resulting from
the refining of the manufacturing process has
resulted in improved levels of antisickling hemo-
globin.27 Together, these protocol improvements
are anticipated to reduce proliferative and hema-
topoietic stresses and therefore may contribute
to reducing the risk of acute myeloid leukemia
after transplantation in patients in Group C. Al-
though follow-up was limited (median duration,
20.9 months [range, 8.5 to 43.1] as of July 26,
2021), no cases of malignant disease have been
reported in Group C patients to date.
In summary, acute myeloid leukemia devel-
oped in a patient who had received LentiGlobin
for sickle cell disease as part of the initial cohort
(Group A) of the HGB-206 study; the develop-
ment of this malignant condition was most
likely independent of insertional oncogenesis.
We have suggested possible contributing factors
to the development of acute myeloid leukemia;
however, a higher proportion of transgene-express-
ing cells may eliminate many of them. Longer
follow-up is required to assess the effect of trans-
plant-mediated gene therapy for patients with
sickle cell disease.
Supported by Bluebird Bio. Medical writing support was
provided by Luke K. Burke, Ph.D. (Synergy Medical Communica-
tions, UK) and was funded by Bluebird Bio.
Disclosure forms by the authors are available with the full
text of this article at NEJM.org.
nejm.org January 13, 2022
 Brief Report

We thank the staff at HGB-206 clinical site for their important
contributions to the care of the patient described in this article;
members of the Bluebird Bio team: Alberto De Iaco (former em-
ployee), Brandon Gentile, Brandon V. Nguyen, Chetanya Pandya
(former employee), Christopher Mucci, Cynthia Rogers, James
Rottman (former employee), Katie Groglio (former employee),
Lauryn M. Christiansen, Madhumita Mahajan (former employ-
ee), Maggie Chen (former employee), Mauris Nnamani (former
employee), Megan D. Hoban, Nick Fenger, Nick Rouillard (for-
mer employee), Noel Namai (former employee), Omar Elbeta-
nony (former employee), Russell W. Goetze (former employee),
Ryan Leenay, Shelby LaBarre (former employee), Stephanie
Hintzen-Matoian, Swathi Krishnan (former employee), Tiffany
Hu (former employee), Xinyan Zhang, Suzanne Plezier Jonkheer,
Dennis Kim (former employee), Briana Deary, Natalia Mackow-
iak, Leanne Ianniello, Heidi Elliot, Laura Demopoulos, Frances
Smith, Joyce O’Connell (former employee), Perrine Geoffroy,
and Katherine Lewis for their contributions; Donald B. Kohn
(Broad Stem Cell Research Center, University of California, Los
Angeles), Andreas E. Kulozik (Department of Pediatric Oncol-
ogy, Hematology, and Immunology, University of Heidelberg),
Franco Locatelli (IRCCS Ospedale Pediatrico Bambino Gesù),
Ryotaro Nakamura (Division of Hematology and HCT, City of
Hope National Medical Center), Eli Pasackow (Generation Bio),
Christof von Kalle (National Center for Tumor Diseases, Heidel-
berg and German Cancer Research Center), David A. Williams
(Boston Children’s Hospital and Dana–Farber Cancer Institute,
Harvard Medical School and Harvard Stem Cell Institute), and
John Wagner (Division of Blood and Marrow Transplantation,
Department of Pediatrics, University of Minnesota) for helpful
discussions; and Pamela Y. Chan of Bluebird Bio for writing and
editorial assistance.

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