Running title: Busulfan and Subsequent Malignancy
Text word count: 2479
Abstract word count: 249
Tables: 5
Figures: 1
Abstract:
The incidence of secondary malignancies associated with busulfan
exposure is considered low, but has been poorly characterized. Because
this alkylating agent is increasingly utilized as conditioning prior to
gene therapy in non-malignant hematologic and related disorders, more
precise characterization of busulfan’s potential contribution to
subsequent malignant risk is warranted. We conducted a literature-based
assessment of busulfan and subsequent late effects, with emphasis on
secondary malignancies, identifying publications via PubMed searches and
selecting those reporting at least 3 years of follow-up. We identified 8
pediatric and 13 adult publications describing long-term follow-up in
570 pediatric and 2,076 adult hematopoietic cell transplant (HCT)
recipients. Secondary malignancies were reported in 0.5% of pediatric
HCT recipients, with no cases of myelodysplastic syndrome (MDS) or acute
myelocytic leukemia (AML). Fatal secondary malignancies were reported in
0.8% of 1887 evaluable adult HCT recipients, and an overall incidence
of secondary malignancies of 4.8% was reported in a subset of 389
evaluable adult patients. We also reviewed long-term results from 8
publications evaluating lentiviral- and human promotor-based
HSC-targeted gene therapy in 215 patients with non-malignant conditions,
in which busulfan/treosulfan monotherapy or busulfan/fludarabine was the
only conditioning. Two malignancies were reported in patients with
Sickle Cell Disease (SCD), one of which was potentially
busulfan-related. No additional malignancies were reported in 173
patients with follow-up of 5-12 years. The incidence of busulfan-related
secondary malignancies is low, and likely to be substantially less than
1% in pediatric transplant recipients, especially those receiving
busulfan monotherapy for non-malignant conditions other than SCD.
Introduction:
The alkylating agent busulfan has been a core component of conditioning
regimens enabling hematopoietic cell transplantation (HCT) prior and
subsequent to its approval in 1999-2002. Busulfan has been particularly
essential in autologous lentiviral mediated gene therapy for
non-malignant genetic disorders, both for investigational programs and
approved therapies. Busulfan-related short-term toxicities are
well-characterized and include mucositis, seizures and hepatic
veno-occlusive disease. Longer term effects include impaired fertility,
organ damage including bronchopulmonary dysplasia, and cancer
predisposition. The incidence of some indolent effects, particularly
regarding fertility in pediatric recipients, has not been precisely
characterized, and is confounded because of the co-administration of
additional cytotoxic therapies in the majority of HCT settings. For
these same reasons, the risk of busulfan-associated secondary malignancy
has also been incompletely delineated. The recent identification of
non-insertional myeloid malignancy in a 45 year old patient with Sickle
Cell Disease (SCD) who had received autologous HSC-directed gene therapy
three years previously resulted in additional discussion of busulfan as
a potentially carcinogenic and leukemogenic agent. As the number of
LV-mediated gene therapy investigational programs and approved therapies
continue to expand, often with compelling efficacy, we believed it was
imperative to enable a more optimal characterization of malignancy
potential in patients receiving this agent, in order to optimally inform
patients and clinicians considering gene therapy options for
non-malignant hematologic and related disorders. This is of particular
importance in pediatric settings. We conducted a literature-based
evaluation regarding the incidence and nature of busulfan-related and
other late effect post-HCT malignancies.
Methods:
A systematic literature search (SLR) was based on the PICOS strategy
(Patient, Intervention, Comparator, Outcome, and Study design) outlined
in Table 1.
EMBASE and MEDLINE (via Proquest Dialog) were searched for the
terms busulfan (including variants), transplantation, conditioning, and
hematopoietic stem cell transplantation (detailed search strategy is
described in supplementary methods) encompassing the publications, in
English language from 2012 through September 2022.
Literature was extracted to a data extraction template (DET) and
duplicates were removed. The entire list was then screened by
title/abstract to identify articles for inclusion/exclusion in the SLR.
Exclusion criteria consisted of conference abstracts, reviews without
patient outcomes, and out of scope publications (conditioning regimens
without Busulfan comprising at least one of the components).
The search results, specific criteria employed, and publications
selected for detailed review are depicted in Figure 1 ,
culminating in cohorts of publications describing results in pediatric
or young adult transplant studies with at least 5 years of median
follow-up, adult transplant studies with at least 5 years of median
follow-up, and adult transplant studies with at least 3 years (but less
than 5 years) median follow-up.
Results:
From the 455 records identified and extracted, only 4 papers were
excluded at the abstract level and 396 when assessing full text. Because
alkylating agent-mediated myeloid malignancies predominantly arise 4-7
years following exposure,, we placed particular
emphasis on studies providing long-term results (median follow-up of at
least 3 years), in both pediatric (or mixed pediatric/adult) or adult
settings. We also evaluated results, with an emphasis on secondary
malignancies, from publications detailing autologous LV-based gene
therapy clinical trials utilizing self-inactivating LVs incorporating
human promotors in hematologic and related disorders, also with emphasis
on studies for which at least 5 years of median follow-up is available.
Excluded from this evaluation are long-term results from LV-based gene
therapy studies in cerebral adrenoleukodystrophy (CALD), a program using
a vector containing a viral promotor associated with in vitromoderate transactivating capability, and the only LV-based program
to-date associated with insertional mutagenesis.
49 papers were then comprehensively assessed based on the previous
inclusion criteria and 25 were excluded for: a) Non-hematologic
pediatric condition; b) Case-controlled risk-analysis; c) Pediatric
study <3y follow-up; d) Adult study < 3y follow-up
and e) Malignancy incidence not evaluable. A list of 24 records were
included for comprehensive review, categorized as follows:
Pediatric/young adult study ≥3y follow-up, n=8 (≥ 5y: n=8); and Adult
study ≥ 3y follow-up, n=16 (≥ 5y: n=8; ≥3y and <5y: n=8).
Eight allogeneic HCT publications were identified describing long-term
outcomes in 642 predominantly pediatric patients, including median
follow-up of at least 5 years in 602 patients, as detailed inTable 2. No secondary malignancies were reported in 6 of these
studies (involving n=504 patients). In one study, 3 of 66 (4.5%)
pediatric leukemia patients receiving busulfan-based multi-agent
conditioning regimens developed secondary malignancies, specifically
thyroid (n=1), basal cell skin cancer (n=1) and meningioma (n=1), in
contrast to 20 of 174 (11.5%) of patients receiving TBI-based regimens
without busulfan. In one additional study, a single patient developed a
secondary melanoma but it was not specified whether this patient had
received busulfan-based (n=72 patients) or TBI-based (n=77)
conditioning. Thus, for 570 evaluable pediatric patients receiving
busulfan-based conditioning, secondary malignancies were reported in 3
patients, representing a 0.5% incidence. There were no reported cases
of AML/MDS in the pediatric population. These findings are in contrast
to secondary malignancies occurring in 131 of 2721 (4.8%) of pediatric
patients receiving TBI-based regimens in the absence of busulfan.
Of note, in one study evaluating long-term effects in very young
pediatric patients (age <2y; 82% with non-malignant
conditions) receiving multi-agent busulfan-based conditioning between
1993 and 2008 (predominantly busulfan, cyclophosphamide and ATG), late
effects included dental complications (92%), short stature (56%),
cognitive deficits (54%) and pubertal abnormalities (28%). In one
study evaluating HCTs in 99 pediatric β-thalassemia patients receiving
transplant between 1985-2012, detailed fertility effects were reported
as follows: 12 of 33 (33%) pre-pubertal females experienced normal
puberty with younger age at HCT (median 2.5 years) relative to those who
had delayed puberty (median age 8.7 years). Pre-pubertal males
experienced subsequent normal puberty in 18 of 22 (82%) of instances.
Eleven of 27 (40%) of evaluable females had at least one successful
pregnancy; and 4 of 21 (19%) of evaluable males fathered at least one
child. Information regarding percentages within groups actively seeking
pregnancy/fatherhood was not provided. A majority of these patients
received either oral busulfan or intravenous therapy prior to the
implementation of pharmacokinetically guided therapy.
Thirteen publications in adult hematologic malignancies were identified
describing long-term results for busulfan/chemotherapy combination
pretransplant conditioning regimens, encompassing 2,076 patients. Three
additional publications described long-term results in adults (also with
hematologic malignancies) when busulfan was combined with TBI and
additional chemotherapy agents, involving 744 patients. Five of the
busulfan/chemotherapy publications and each of the three
busulfan/TBI/chemotherapy publications involved median follow-up
exceeding 5 years, summarized in Table 3 ; the remaining 8
selected studies involved median follow-up of between 3 and 5 years,
summarized in Table 4 .
For 11 of the 13 studies evaluating busulfan/chemotherapy conditioning
regimens, long-term outcomes were reported as fatal secondary
malignancies, involving 1,887 patients. (It is presumed that these
malignancies, resulting in patient deaths, are identified in contrast to
localized or less aggressive secondary neoplasms such as non-invasive
skin cancers, which were reported in >5% of patients in
some adult series). Incidences of fatal secondary malignancies ranged
from 0 to 5.9% across studies (median incidence 0.7%). Overall there
were 16 fatal secondary malignancies reported subsequent to HCT in 1,887
patients, representing a 0.8% incidence. Of note, of the four studies
with the highest incidence of fatal secondary malignancies, 3 of these 4
employed reduced intensity (RIC) conditioning, as did 2 of the 4 studies
with the lowest incidences.
The overall incidences of secondary malignancies were reported in four
of the adult studies, involving 389 patients. Nineteen of these 389
(4.8%) patients encountered secondary malignancies, with the incidence
ranging between 0 and 10.7%. Importantly, 16 of the 19 secondary
malignancies were reported from a single study in high-risk hematologic
malignancies in an older adult population (median age 61 years, 33%
above 65 years at time of HCT). Malignancies in this study included one
of the previously mentioned fatal cancers, and 15 additional secondary
malignancies (7 of which were dermatologic); 14 of these 15 secondary
malignancies occurred in patients who had experienced either acute (n=6)
or chronic (n=8) GVHD.
The highest incidences of secondary malignancies occurred in the three
series in which busulfan and additional chemotherapy agents were
administered in conjunction with TBI, as conditioning for hematologic
malignancy-directed HCTs in adults. Secondary malignancy incidences in
these three studies, involving 744 patients, were 6%, 7.5% and 8%
respectively. Specific malignancies were described in two of these
publications and were non-invasive skin cancers, occurring in a total of
three patients.
The potential contribution of busulfan to secondary malignancies is
potentially best delineated by the experience in autologous
hematopoietic stem cell directed gene therapy studies, several of which
utilize busulfan monotherapy or combination exclusively with
fludarabine. In these studies, busulfan dosage ranged from reduced
intensity (RIC; as low as AUC 10-19 mg*h/L) to myeloablative (70-85
mg*h/L). Long-term follow-up results are available for several of these
trials, with follow-up durations of 5-12 years available across 8 trials
encompassing 173 patients, detailed in Table 5. Also described
in Table 5 are results from 42 patients enrolled in SCD studies
for whom follow-up of at least 3 years is available. Notably, the only
malignancies occurring in these studies were in the SCD studies, in two
patients receiving therapy as part of the initial Group A process,
involving bone marrow harvest, lower CD34+ cell doses, and more limited
hemoglobin response relative to the more recently treated 35 patients.
Both myeloid malignancies harbored mutations in oncogenes RUNX1 and
PTPN11, and monosomy 7 cytogenetic abnormalities. In one case, the
malignant cell population was comprised of gene-corrected cells, with an
integration in proximity to VAMP4 , a loci not consistent
with mutagenic potential. In the second case, no gene markings were
present in the malignant population, and the AML was considered
potentially secondary to busulfan in the context of underlying SCD.
Importantly, for the entirety of LV-based gene therapy studies utilizing
vectors with human promotors, no malignancies have been reported
secondary to insertional mutagenesis. A recent update indicated no
clonal hematopoiesis or associated detectable mutations in 10 ADA-SCID
gene therapy recipients who received RIC busulfan with 7-10 years of
follow-up.
Discussion:
In summary, the incidence of busulfan-associated secondary malignancies
is low, particularly in pediatric settings, with a majority of long-term
evaluations indicating an incidence less than 1%, and an overall
aggregate incidence across more than 500 patients of 0.5%. Secondary
MDS/AML in the setting of pediatric busulfan exposure was not reported
and is likely to be rare. The incidence of post-busulfan secondary
malignancies was higher in adult transplant studies, but also relatively
limited. The incidence of fatal secondary malignancies is likely lower
than 1%, and the overall incidence of secondary malignancies is likely
lower than 5%, including a substantial proportion of non-invasive
dermatologic malignancies. A substantial proportion of the secondary
malignancies identified were identified in studies with extensive
participation of older adults. It is likely that older age confers a
higher susceptibility to conditioning-related malignancies, consistent
with this association in more general populations. Results in adult
populations are unlikely to precisely inform potential malignant risks
for pediatric patients.
Notably, in the adult transplant series, secondary malignancies were
reported both in cohorts receiving myeloablative and RIC regimens. It is
likely that although RIC regimens involve an overall reduced cytotoxic
burden, this effect is potentially countered by survival of
hematopoietic progenitors which have been exposed to alkylator and other
therapies with potential for mutagenic DNA damage. RIC regimens may also
be more likely utilized in older adult cohorts, who are at higher
baseline cancer risk.
A limitation of this study is that the overwhelming proportion of both
allogeneic and autologous HCTs were performed in settings of hematologic
malignancies, in which multi-agent conditioning regimens were likely to
have been preceded by induction and additional chemotherapies, such that
patients’ overall exposure to cytotoxic therapy was extensive. This
limits the extent to which one may ascribe the specific contribution of
busulfan to secondary malignancies or other long-term complications.
These results also pre-date the implementation of therapeutic drug
monitoring (TDM) to enable more precise busulfan dosing; because the
pharmacokinetics of oral and intravenous busulfan are highly variable,
there was higher potential for overexposure in these earlier studies
than in current programs. An additional limitation is that very few
publications provided detailed results regarding other long-term effects
of interest – including fertility and neurocognitive and overall
development in pediatric transplant recipients. These remain substantial
risks for children receiving high-dose combination chemotherapy; as with
secondary malignancies, the precise risks conferred by busulfan cannot
be determined because of the multi-agent nature of the regimens
evaluated. Long-term results from an EBMT dataset are likely forthcoming
and will hopefully provide more precise information regarding fertility
impairment in adult survivors of pediatric transplant.
The most busulfan-specific results are likely to emanate from autologous
HSC-directed gene therapy studies, including several in which busulfan
was utilized exclusively as a conditioning agent (at exposures ranging
from AUC 10-85 mg*h/L). As indicated, long-term results from the
earliest LV-mediated studies are now available, with 5-10 years of
follow-up for more than 150 patients. In these settings, administration
of busulfan conditioning therapy is less confounded by concomitant
conditioning agents or prior anti-cancer therapies. The only potential
busulfan-related malignancy (AML) developed in a patient with SCD, a
condition increasingly recognized as predisposing to myeloid malignancy,
albeit with uncertainty regarding the specific contributing factors or
overall increase in risk. Follow-up of more than 5 years in patients
receiving busulfan-based conditioning for genetic disorders other than
SCD indicates that the risk of secondary malignancy is likely to be very
rare.