REVIEwS

Advancing therapy for osteosarcoma

Jonathan Gill    and Richard Gorlick    ✉
Abstract | Improving the survival of patients with osteosarcoma has long proved challenging,
although the treatment of this disease is on the precipice of advancement. The increasing
feasibility of molecular profiling together with the creation of both robust model systems and
large, well-​annotated tissue banks has led to an increased understanding of osteosarcoma biology.
The historical invariability of survival outcomes and the limited number of agents known to be
active in the treatment of this disease facilitate clinical trials designed to identify efficacious novel
therapies using small cohorts of patients. In addition, trial designs will increasingly consider the
genetic background of the tumour through biomarker-​based patient selection, thereby enriching
for clinical activity. Indeed, osteosarcoma cells are known to express a number of surface proteins
that might be of therapeutic relevance, including B7-​H3, GD2 and HER2, which can be targeted
using antibody–drug conjugates and/or adoptive cell therapies. In addition, immune-​checkpoint
inhibition might augment the latter approach by helping to overcome the immunosuppressive
tumour microenvironment. In this Review, we provide a brief overview of current osteosarcoma
therapy before focusing on the biological insights from the molecular profiling and preclinical
modelling studies that have opened new therapeutic opportunities in this disease.

Despite being the most common primary bone cancer in
children and young adults, osteosarcoma is a very rare
cancer, with approximately 400 new cases diagnosed
annually in children and young adults in the USA1,2.
The incidence of the disease peaks in adolescence
(4.4 cases per million individuals)1,2, which coincides
with the pubertal growth spurt. Osteosarcoma most
commonly arises in the metaphysis of the long bones
near the growth plates, with two-​thirds of tumours
emerging around the knee joint in the distal femur, fol-
lowed by the proximal tibia as the second most frequent
site; the proximal humerus is the third most common
site, accounting for 10% of tumours3. More than 85% of
patients present with localized disease. Of the patients
who present with metastatic disease, 74% present with
lung-​only metastases, 9% with bone-​only metastases,
and 8% with both bone and lung metastases3. A second
peak in the incidence of osteosarcoma occurs in adults
aged >65 years (4.2 cases per million individuals), often
manifesting as secondary cancers (in 25% of patients) or
as a consequence of Paget disease (in 10%)1. This Review
is focused primarily on the treatment of osteosarcoma in
adolescent and young adult patients. The rarity of this
Division of Pediatrics, cancer has necessitated collaborative efforts involving
The University of Texas MD cooperative groups, such as the Children’s Oncology
Anderson Cancer Center,
Houston, TX, USA.
Group (COG) in the USA, as well as international
✉e-​mail: rgorlick@ collaborations to complete clinical trials.
mdanderson.org The standard therapy for osteosarcoma, comprising
https://doi.org/10.1038/ surgery and chemotherapy, was established in the 1980s
s41571-021-00519-8 and results in long-​term survival in >60% of patients
presenting with localized disease4; however, limited
therapeutic progress has been made since that time. An
increased understanding of the biology of the disease
has highlighted its heterogeneity and revealed molecu-
lar aberrations that define potential patient subgroups.
These advances were made possible by the creation of
well-​annotated tissue banks beginning at the turn
of the century in parallel with the broader availability of
technologies for comprehensive molecular profiling.
Many of the most common genetic alterations
detected in osteosarcomas, such as TP53 break-​apart
translocations and RB1 deletions that abrogate the
expression of key tumour-​suppressor proteins, have
been challenging to target therapeutically. Nevertheless,
small subsets of these tumours harbour potentially
targetable somatic alterations. More broadly, clinical
trial designs incorporating biomarker-​based selection
might increase the proportion of patients with oste-
osarcoma who benefit from a particular treatment.
Cellular differentiation within the mesenchymal cell
lineage is a characteristic feature of osteosarcoma and
results in the consistent expression of several cell-​surface
molecules. Accordingly, novel antibody-​b ased and
adoptive cell therapy approaches are broadening the
range of possible therapeutic targets in osteosarcoma;
immune-​checkpoint inhibitors (ICIs), although disap-
pointing as single agents in this disease, might have roles
in enhancing the efficacy of such approaches.
Notably, robust preclinical models of osteosarcoma
are now available and could potentially facilitate drug

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Key points
• Osteosarcoma is the most common primary malignant tumour of bone, with a peak
incidence in adolescents and young adults coinciding with the pubertal growth spurt.
• Limited progress has been made in improving the survival outcomes in patients with
osteosarcoma over the past four decades.
• Improved molecular characterization has revealed subcategories of osteosarcoma
that might enable a precision medicine approach with agents targeting key
alterations in a particular pathway.
• Tumour-​suppressor genes are commonly altered in this disease, particularly TP53
(>90%) and RB1 (30%). Molecular targets include receptor tyrosine kinases, CDK4/6,
Aurora kinase B and DNA damage response pathways.
• Immune-​based targeted therapies, including monoclonal antibodies, antibody–drug
conjugates and chimeric antigen receptor T cells targeting cell-​surface proteins
commonly overexpressed in osteosarcoma, are in active clinical development.
• Owing to advances in biological understanding, the development of robust
preclinical models, the feasibility of rapid clinical testing and novel treatment
concepts, long-​awaited improvements in the outcomes of patients with
osteosarcoma are anticipated in the near future.
development. Moreover, the standardization of trial
designs and efficacy end points has ensured that mul-
tiple groups are capable of conducting clinical studies
rapidly and efficiently. These advances suggest the field
is on the verge of progress in the treatment of osteosar-
coma. Hence, advances in osteosarcoma therapy serve
as the organizing principle of this Review, encompassing
current therapy, targeted treatments based on specific
molecular alterations and immune-​based approaches.
Current osteosarcoma therapy
The established three-​drug chemotherapy regimens
used in the treatment of localized osteosarcoma has
led to consistent patient outcomes over the past four
decades. In North America, the most common chemo-
therapy regimen, MAP, consists of high-​dose methotrex-
ate, doxorubicin (adriamycin) and cisplatin (platinol)
administered both before and after definitive surgical
resection. In the field of osteosarcoma, the terminology
of ‘neoadjuvant’ and ‘adjuvant’ chemotherapy has been
replaced with ‘induction’ and ‘consolidation’ treatment,
respectively. Numerous clinical trials have tested vari-
ous combinations of the five chemotherapeutic agents
known to be active in this disease (methotrexate, doxo-
rubicin, cisplatin, ifosfamide and etoposide), some using
pathological response at the time of definitive surgery to
select consolidation therapy, without additional progress
being made5–9. These studies have revealed that frontline
agents can be replaced with other active drugs but that
adding more agents into the initial treatment regimen
does not improve outcomes (Fig. 1) nor does adjusting
therapy based on pathological response.
In patients with relapsed and/or metastatic osteosar-
coma, surgery with metastasectomy has been shown to
provide survival benefit10–12. Chemotherapy regimens
comprising ifosfamide and etoposide or gemcitabine
and docetaxel have some efficacy in patients with unre-
sectable relapsed disease13–15. Data from several phase II
trials of various treatments demonstrate the dismal
outcomes of patients with relapsed, unresectable oste-
osarcoma, with an aggregate event-​free survival (EFS)
of only 12% at 4 months16; however, this situation
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creates opportunities to rapidly evaluate novel thera-
pies in single-​arm clinical trials using small numbers
of patients. The outcomes of patients with resectable
recurrent disease (typically pulmonary metastases) are
also highly conducive to single-​arm study designs, with
a median EFS of 4 months and a 2-​year EFS of 12%17.
Genomic alterations and heterogeneity
Since the advent of karyotyping, osteosarcomas have
been known to be genomically complex18,19. Even before
analyses of chromosomes were widely performed, rou-
tine histological staining revealed substantial variation in
the appearance of individual tumours, with the pattern
of cellular differentiation defining the disease subtype20.
Conventionally, osteosarcomas are classified as osteo-
blastic (50%), chondroblastic (25%) or fibroblastic (25%)
according to their predominant differentiation pattern.
These classifications are somewhat arbitrary given that
the majority of tumours contain more than one histo-
logical pattern, which suggests considerable intra-​patient
heterogeneity21. The frequent occurrence of osteosar-
coma in patients with cancer predisposition syndromes,
such as Li–Fraumeni syndrome and hereditary retino-
blastoma, led to the early recognition of prevalent alter-
ations in tumour-​suppressor genes in this malignancy,
including TP53 (>90%) and RB1 (30%)22. The identifi-
cation of these and other recurrently altered genes raised
hopes that certain molecular alterations crucial to the
pathogenesis or drug sensitivity of osteosarcoma would
serve as prognostic or predictive biomarkers. In numer-
ous paediatric malignancies, including neuroblastoma
and acute lymphoblastic leukaemia, patients receive
risk-​stratified treatment, often aided by the identification
of prognostic molecular characteristics, such as MYC
amplification or the Philadelphia chromosome translo-
cation, respectively23,24. With this goal in mind, numer-
ous institutional osteosarcoma tissue banks began to be
created and, in 1998, a national biobank was established
in the USA by the COG. The COG Tissue Bank has been
described in several publications25,26, which highlight the
broad availability of osteosarcoma samples as well as
the philanthropic support of advocacy groups such as the
QuadW Foundation, which was a major contributor to
the success of the initiative. Numerous other organiza-
tions around the world have initiated biobanks including
osteosarcoma, such as the Bone Cancer Research Trust
in the UK and EuroBoNeT in the European Union. With
the continuous evolution of tissue banking, efforts do not
have to be centred in a single institution. For example, the
Count Me In initiative led by the Broad Institute of MIT
and Harvard (with institutional review board approval
as an entity) aims to obtain tissue samples and data from
patients directly and includes a specific Osteosarcoma
Project component. These tissue banks have facilitated a
large number of molecular profiling studies of osteosar-
coma, predominantly focused on limited sets of genes27,
although, unfortunately, a reproducible prognostic factor
has not been identified thus far.
Patterns in data emerging from comprehensive
molecular profiling studies have prompted a paradigm
shift towards broader analyses of pathways and genomic
signatures, rather than of individual genes, to identify
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100
Mean EFS with
four-drug regimens157
COSS-86 (MAPI)158 90
IOR/OS-4 (MAPI)159
POG-8651 (MAP BCD)160
INT-0133 (MAPI)161 80

ISG-SSG-1 (MAPI) 162

EOI-80861 (AP V BCD)163

70

Mean EFS with

three-drug regimens157
60
INT-0133 (MAP)161
MIOS (MAP)164
EFS (%)

MEI165 50
API8
COSS-80 (MAP)166
40
COSS-82 (MAP)167
EOI-80831 (MAP)168
SFOP-OS94 (MEI)169 30
IOR/OS-2 (MAP)170
IOR/OS-3 (MAP)170
SSG-VIII (MAP)171 20

Mean EFS with

two-drug regimens157 10
EOI-80861 (AP)163
EOI-80931 (AP)163
0
SFOP-OS94 (MA)169 0 1 2 3 4 5
Follow-up duration (years)

Fig. 1 | EFS with chemotherapy regimens comprising two, three or four drugs in patients with osteosarcoma.
The dashed lines depict the event-​free survival (EFS) curves from numerous large-​cohort trials of various chemotherapy
regimens in patients with osteosarcoma. The aggregate EFS curves of trials investigating combinations of two, three
or four cytotoxic agents are shown using red, blue and green bold lines, respectively. Notably, the green EFS curves
corresponding to four-​drug regimens overlap substantially with the blue lines relating to three-​drug regimens. Thus,
the outcomes of patients treated with various three-​drug or four-​drug regimens are effectively the same; however,
patients treated with two-​drug combinations have inferior outcomes (red lines). A, doxorubicin; B, bleomycin;
C, cyclophosphamide; D, dactinomycin; E, etoposide; I, ifosfamide; M, methotrexate; P, cisplatin; V, vincristine157–171.

pathogenetic aberrations28–33. In a seminal publication
describing chromothripsis34, whereby chromosomal
shattering results in numerous simultaneous genomic
rearrangements, osteosarcoma was identified as one of
the tumour types in which this phenomenon occurs.
Indeed, chromothripsis is now recognized as a com-
mon oncogenic event in osteosarcomas — detectable
in up to 75% of tumours — and can involve multiple
chromosomes34,35. The loss of an organized chromosome
structure has complicated the identification of genetic
commonalities across osteosarcomas, with multiple pub-
lished sequencing studies revealing similar findings but
focusing on different perturbed pathways or genomic
signatures. For example, some groups have focused on
altered tumour-​suppressor genes22,29,30,36, some on par-
ticular signalling pathways31,32 and others on the immune
landscape of the disease28,33,37.
In a whole-​genome sequencing (WGS) study, differ-
ences in the number and location of structural altera-
tions were observed across most osteosarcomas, with
a few shared structural variations22. However, certain
genes were commonly altered, albeit through different
somatic alterations, including the tumour-​suppressor
genes TP53 (95%), RB1 (29%), ATRX (52%) and
DLG2 (52%)22. In addition to the structural alterations
observed, kataegis was observed in 50% of the samples22.
A whole-​exome sequencing (WES) study revealed alter-
ations in the same genes, although the unique mutations
identified in each sample underscore the substantial
interpatient heterogeneity of this disease29. In a separate
WES study30, a genomic instability signature character-
istic of BRCA1/2-deficient tumours was observed in 80%
of osteosarcomas.
With regard to specific signalling pathways, altera-
tions in IGF1, IGF1R, IGF2R, IGFBP5 or components of
the downstream signalling pathway have been detected
in 27% of 112 osteosarcomas through WGS or WES,
with high-​level IGF1R amplifications (≥15 copies)
detected by fluorescence in situ hybridization in 14% of
an additional 87 samples31. In another study32, pathway

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analyses and other computational or experimental
approaches were used to decipher the heterogeneity
of aberrations observed in complementary WGS, WES
and RNA sequencing data from patients with osteosar-
coma. The results indicate the convergence of unique
alterations on the activation of the PI3K–ATK–mTOR
pathway32. Combined genomic and transcriptomic
analyses have also enabled the identification of multiple
fusion transcripts that only rarely correlate directly with
chromosomal rearrangements in osteosarcoma sam-
ples, suggesting the involvement of post-​transcription
trans-​splicing36. These fusion transcripts, although
involving multiple loci in different cell lines and
patient samples, most commonly affect the TP53 locus
and serve as another mechanism by which TP53 is
inactivated in osteosarcoma36.
A number of studies have evaluated the immuno­
genomic landscape of osteosarcoma. The resulting data
demonstrate that osteosarcomas can have an immune
‘hot’ or ‘cold’ signature or an intermediate level of
immune cell infiltration33. Most of the cold tumours
demonstrated a substantial number of genes with copy
number losses and decreased expression of HLA genes,
leading to immune escape37. The hot subset demonstrated
an adaptive immune resistance mechanism involving
upregulation of T cell inhibitory immune-​checkpoint
proteins, including PD-​L1 and CTLA4 (ref.37). Notably,
publicly available deep-​sequencing data from 88 patients
with osteosarcoma, which were generated as part of the
US National Cancer Institute (NCI) Therapeutically
Applicable Research to Generate Effective Treatments
(TARGET) Osteosarcoma project, have been used to
generate a signature comprising 13 immune-​related
genes that are prognostic of distant metastasis and
overall survival (OS)28.
In public presentations, TARGET Osteosarcoma pro-
ject investigator Paul Meltzer has made the analogy that
the mutational profiles of osteosarcomas are compara-
ble to snowflakes38: like snowflakes, all osteosarcomas
share a common recognizable ‘shape’ but each tumour is
unique. Taken together, however, these studies demon-
strate how an explosion of knowledge is yielding an
understanding of common patterns visible through the
enormous genomic complexity of osteosarcoma.
Subcategorization of osteosarcomas
In a landmark study, the Sweet-​C ordero group 39
attempted to establish some organization in the inter-
patient heterogeneity of osteosarcoma by defining dis-
ease subclassifications based on pathway alterations.
Using deep-​sequencing to define genomic alterations,
osteosarcoma could be subclassified into six groups
according to the key signalling pathways activated:
cyclin E1/CDK2, MYC/CDK9, CDK4/CDK6/FOXM1,
PTEN/PI3K/AKT1/mTOR, AURKB and VEGFA/
VEGFR39 (Fig. 2). While conceptually deriving from
approaches taken in classifying other cancer types, such
as medulloblastoma40, it must be acknowledged that
more overlap occurs in the context of osteosarcoma. In
an analysis of patient-​derived xenograft (PDX) models
developed by the NCI Paediatric Preclinical Testing
Consortium (PPTC), for example, some tumours were
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found to have multiple pathway alterations that could be
categorized into more than one group, whereas others
were not easily classified into any of the groups (R.G.,
unpublished data) (Fig. 2). On the whole, the genomic
complexity and heterogeneity observed in multiple
studies indicate that osteosarcoma might need to be
considered as more than one disease.
Models of osteosarcoma
Over the past 15 years, >100 drugs have been screened for
activity in osteosarcoma using PDX models developed
by the PPTC41 (Figs 2,3; Supplementary Table 1). More
recently, a European consortium has established the
Innovative Therapies for Children with Cancer Paediatric
Preclinical Proof-​of-​c oncept Platform (ITCC P4),
which is seeking to establish and fully characterize 400
new PDX models of high-​risk paediatric solid tumours,
including osteosarcomas. Indeed, in the aforemen-
tioned study by the Sweet-​Cordero group39, common
osteosarcoma-​associated molecular alterations were
accurately recapitulated in a set of PDX models and
responses to genome-​informed treatments were seen in
all PDXs tested. The PPTC have also sequenced and char-
acterized 31 PDX models of osteosarcoma, demonstrat-
ing substantial tumour heterogeneity that recapitulates
the high prevalence of complex genomic rearrangements
described in patients with osteosarcoma42.
Despite sharing the same genomic alterations as
their human tumour counterparts, PDX models have
clear limitations. In particular, therapeutic activity in
these models does not necessarily translate into efficacy
in clinical trials (Fig. 2). For example, both glembatu-
mumab vedotin (a transmembrane glycoprotein NMB
(GPNMB)-​targeted antibody–drug conjugate (ADC)
with a microtubule inhibitor payload) and the cyto-
toxic agent eribulin (also a microtubule inhibitor) were
predicted to have activity against osteosarcoma based
on PDX modelling (Fig. 3) but ultimately had limited
efficacy in patients with relapsed osteosarcoma43,44. In
the case of eribulin, this disparity probably reflects a
failure to fully account for pharmacokinetic differences
between mice and humans45. A major concern is that
PDX tumours are necessarily implanted in immunode-
ficient mice; therefore, these models fail to recapitulate
the immunological aspects of cancers and cancer treat-
ments, which in particular poses substantial challenges
to the testing of immunotherapies. Defining the level of
activity in PDX models that translates into efficacy in
clinical trials is also problematic: should this be deter-
mined by the proportion of models demonstrating a
response or by the degree of response within a single
model? This question is confounded by the fact that the
lack of activity of any novel agents tested in patients to
date limits the ability to extrapolate a positive predictive
value from the preclinical models.
In addition to PDX models, numerous transgenic
models of osteosarcoma have been generated but,
notably, have rarely been used for drug testing. These
transgenic models include Tp53 and combined Tp53
and Rb1 knockouts driven by an osteoblast-​based pro-
moter, which result in osteosarcomas in 77% and 100%
of mice, respectively46,47. One potential flaw of these
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a e
MYC activation VEGF VEGF–VEGFR
pathway alterations
Predicted Response
responder observed Predicted Response
PIP2 PIP3 responder observed
OS1 c PI3K
P P
OS2 P P OS1
CDK4 or FOXM1
upregulation OS2
OS9 VEGFR
Cyclin D PTEN AKT OS9
OS17 Predicted Response
CDK4 responder observed OS17
OS31 d PTEN–PI3K–AKT
OS1 pathway alterations OS31
OS33
P OS2 Predicted Response OS33
MYC RB1 OS9 responder observed
OS17 OS1
OS31 OS2
OS33 OS9
OS17
FOXM1
Cyclin E1 OS31
f
CDK2 OS33
b Overexpression of
CCNE1 amplification AURKA or AURKB
Predicted Response Predicted Response
responder observed responder observed
OS1 OS1
Aurora A
OS2 OS2
OS9 Aurora B OS9
OS17 OS17
OS31 OS31
OS33 OS33

MCR CR PR SD PD2 PD1 Not tested

Fig. 2 | Molecular categories of osteosarcoma and responses to the
corresponding molecularly targeted treatments in PPTC PDX models.
The National Cancer Institute Paediatric Preclinical Testing Consortium
(PPTC) patient-​derived xenograft (PDX) models of osteosarcoma (see
Supplementary Table 1) that are predicted to respond to specific targeted
therapies based on their characteristic genomic alterations are indicated
in green in the first column of each table. The actual responses observed in
each of the PDX models are indicated in the second column of each table.
a | Two models (OS2 and OS17) have activation of MYC signalling and are
therefore predicted to respond to agents targeting this pathway, although
such agents have not yet been tested by the PPTC. b | Three models (OS1,
OS9 and OS17) were predicted to respond to CDK2 inhibitors, owing to
CCNE1 amplification; however, activity was observed only in the OS1
model in the form of SD. c | Two models (OS1 and OS17) have upregulation
of CDK4 and/or FOXM1 and are therefore predicted to respond to CDK4/6
inhibitors, which have not been tested by the PPTC to date. d | PTEN loss or
activating AKT1 mutations in three models (OS1, OS9 and OS17) were
predicted to confer sensitivity to the inhibition of the PI3K–AKT–mTOR
signalling pathway, although the AKT inhibitor MK-2206 had minimal or
no activity in these models. e | The one model predicted to respond to
agents targeting the VEGF–VEGFR signalling pathway (OS17) was indeed
responsive to VEGFR-​directed tyrosine kinase inhibitors. f | In addition, SD
was observed in a model (OS33) that was not expected to respond. Two
models (OS2 and OS33) were predicted to respond to the Aurora kinase
inhibitor MN8237 owing to the overexpression of AURKA or AURKB;
however, responses observed in the PPTC PDX models did not correlate
with these genomic alterations. Note that some models (such as OS1 and
OS17) carry genomic alterations that predict responses to various
inhibitors of multiple pathways, suggesting that combination therapy
might be warranted. CR, complete response (disappearance of measurable
tumour mass for at least one time point); MCR, maintained complete
response (tumour volume <0.10 cm3 at the end of the study period); PD1,
progressive disease (>25% increase in tumour volume at the end of the
study period); PD2, delayed progressive disease (same as PD1 but with
tumour growth delay); PR, partial response (≥50% tumour regression for at
least one time point but with measurable tumour); SD, stable disease
(<50% regression and ≤25% increase in tumour volume at the end of the
study period).

models relates to their propensity for the development
of tumours in the jaw and head, which typically occur in
only 9% of patients with osteosarcoma46,47. A transgenic
model in which the expression of Myc is driven by the
lymphocyte-​specific enhancer EµSRα in the presence
of tetracycline leads to osteosarcomas in 1% of mice,
presumably owing to the activation of the enhancer
element in immature osteoblasts; the resulting tumours
demonstrate oncogene addiction to Myc48. MYC is
overexpressed in 41% of human osteosarcomas and this
feature is associated with an increased risk of metasta-
sis as well as with unfavourable survival outcomes49,50.
Historically, Fos transgenes, as well as radiation and
chronic parathyroid hormone administration, have also
been used to induce osteosarcoma formation in mice51,52.
These various transgenic models enable the evaluation
of osteosarcomas in a native microenvironment, which
mitigates some of the issues associated with PDX models.

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Fig. 3 | Comparisons of the activity of aDCs and drugs similar to the
aDC payloads in PDX models of osteosarcoma. In the National Cancer
Institute Paediatric Preclinical Testing Consortium (PPTC) patient-​derived
xenograft (PDX) models of osteosarcoma (see Supplementary Table 1),
variable responses are observed between antibody–drug conjugates (ADCs)
targeting HER2 (trastuzumab deruxtecan), LRRC15 (samrotamab vedotin;
ABBV-805), GPNMB (glembatumumab vedotin), or B7-​H3 (m276-​PBD) and
unconjugated drugs with comparable mechanisms of actions to the ADC
payloads. This disparity suggests that the activity of ADCs is at least partially
driven by the selective, antibody-​mediated targeting of the drug to
tumours. Nevertheless, some models are resistant regardless of the ADC
target (for example, the OS1 model is resistant to both trastuzumab
deruxtecan and samrotamab vedotin), which might reflect resistance to the
payload of the ADC. Conversely, some models might be less sensitive to
the ADC than the free drug owing to potential differences in the expression
of the target antigen. CR, complete response (disappearance of measurable
tumour mass for at least one time point); MCR, maintained complete
response (tumour volume <0.10 cm3 at the end of the study period); PD1,
progressive disease (>25% increase in tumour volume at the end of the
study period); PD2, delayed progressive disease (same as PD1, but with
tumour growth delay); PR, partial response (≥50% tumour regression for at
least one time point, but with measurable tumour); SD, stable disease (<50%
regression and ≤25% increase in tumour volume at the end of the study
period).

However, transgenic models might still not accurately
recapitulate immune responses to osteosarcomas in
patients owing to the differences between the mouse and
human immune systems. An alternative strategy to over-
come this potential issue involves the creation of PDX
models of osteosarcoma in humanized mice53. This field
is becoming more complex and a range of models with
varying degrees of immune reconstitution are becoming
available54. With humanized PDX models, the haemato-
poietic stem cells used for immune reconstitution should
ideally be derived from the same donor patient to avoid
graft-​versus-​tumour effects. Such models can be used to
evaluate ICIs and other immunotherapies.
Finally, osteosarcomas arise spontaneously in
canines and have similar patterns of DNA alterations
to their human counterpart55,56. Veterinary clinical tri-
als in canines are feasible through comparative oncol-
ogy consortiums sponsored by the NCI and other
organizations57. Such trials bear some of the same issues
as human clinical trials in that the canines require
informed consent from their owners for participation.
However, the owners might have differing thresholds of
acceptance for the toxicities associated with treatment
compared with parents of children with osteosarcoma.
A potential major disadvantage of conducting these trials
is that the cost of the medications is often consigned to
the dog owners.
Preclinical testing of potential targeted therapies
should be considered in the context of pathway alter-
ations present in the PDX models. Owing to the inter-
patient heterogeneity, if administered in an unbiased
fashion across all models, a targeted therapy by defini-
tion will only induce responses in a subset of the mod-
els tested. Thus, genomic characterization of the PDX
models is required to create a responder hypothesis to
enrich for pathway alterations present in specific models.
This process is confounded by the fact that some PDX
models have alterations in multiple pathways (Fig. 2).
Understanding the effects of overlapping pathway alter-
ations and responsiveness to targeted therapies will
require testing with both single agents and combination
treatments in order to identify driver versus bystander
alterations. In addition, the complexity of the pathway
alterations underscores the need to expand the number
of PDX models being utilized in order to better encap-
sulate the scope of the aberrations found in patients.
This issue is further complicated by the fact that most
tumour-​profiling panels include only subsets of genes
that do not encompass the entire pathway associated
with a given alteration. Tumours likely to be suscepti-
ble to a potential targeted therapy might therefore be
missed with the use of targeted gene panels. Hence, the
emerging concept is that precision medicine for patients
with osteosarcoma will require a broader view of cellular
pathway alterations (rather than of specific gene muta-
tions) to guide biomarker-​based clinical trials designed
to maximize the potential benefits of targeted therapies.
Although we have highlighted the weaknesses of each of
the model systems, we want to emphasize that these tools
provide a crucially important starting point for develop-
ing hypotheses regarding which treatments will be active
and should be prioritized for clinical development.
Targeted therapy of osteosarcoma
Tailoring therapy using a biomarker that predicts a
response (or resistance) to a specific molecularly tar-
geted treatment is the central concept of precision med-
icine. In the context of osteosarcoma, the challenges

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facing precision medicine include the facts that most of
the recurrent molecular alterations are difficult to tar-
get therapeutically (for example, those in TP53 or RB1)
and that the alterations constituting dependencies of the
tumour are often unclear on the background of multiple
secondary genomic alterations. Moreover, clinical tri-
als using pathway alterations as biomarkers for patient
selection present a substantial challenge in a rare disease
with approximately 400 patients diagnosed annually in
the USA. However, the COG analysis16 data, revealing
that the outcomes of patients with relapsed, unresectable
osteosarcoma have remained unchanged across different
phase II trials of numerous agents performed over a long
period of time (with an aggregate 4-​month EFS of only
12%), justifies the use of historical control groups. This
situation has increased the feasibility of clinical trials
designed to evaluate the efficacy of novel therapies in
small subsets of patients with this rare disease. In multi-
ple single-​arm phase II trials performed by the COG, the
primary end point has been a 40% disease control rate
(DCR) at 4 months43,44. Any active agents that demon-
strate this level of activity will subsequently be tested
as a randomized addition to standard chemotherapy in
patients with newly diagnosed osteosarcoma. This strat-
egy is predicated on the hypothesis that improvements in
frontline therapy have the highest likelihood of increas-
ing survival, with salvage chemotherapy thus far adding
no obvious benefit beyond surgery alone in patients with
relapsed disease10–12. This strategy has enabled the rapid
completion of clinical studies. For example, the phase II
AOST1322 trial of eribulin in patients with relapsed oste-
osarcoma completed enrolment within a few months44,58.
Furthermore, studies performed worldwide have used
the 4-​month DCR end point, thus facilitating cross-​study
comparisions16. The success of this approach underscores
the potential for studies in biomarker-​selected subsets of
patients. Perhaps, however, some conceptual evolution
of the precision medicine paradigm is needed in osteosar-
coma: rather than identifying direct target–drug relation-
ships that define therapeutic activity, biomarker-​based
selection could be used to increase the probability that
an agent will be efficacious in a heterogeneous patient
population. In its simplest form, this approach might
involve enrichment for patients with high-​risk disease,
with MYC overexpression being a leading candidate
molecular biomarker of prognosis49,50.
Tyrosine kinase inhibitors. Multiple receptor tyro­
sine kinases (RTKs) have been demonstrated to be
expressed in osteosarcoma cells. Most tyrosine kinase
inhibitors (TKIs) cannot necessarily be considered pre-
cision medicines given that they act on multiple kinases
to a variable degree (Supplementary Table 2). Indeed,
clinical trials of TKIs in patients with osteosarcoma to
date have not involved tumour profiling and thus the
results do not provide a clear biological rationale for
the activity observed. Nevertheless, multiple TKIs have
demonstrated efficacy in patients with relapsed and/or
unresectable disease, including sorafenib (4-​month
progression-​free survival (PFS) of 46%)59, regorafenib
(median PFS of 3.6 months versus 1.7 months with
placebo)60, cabozantinib (4-​month PFS of 71%)61,62,
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lenvatinib (4-​month PFS of 33%)63 and pazopanib
(median PFS of 5.5 months in patients with cancers of
mixed histologies, 50% of which were osteosarcomas)64.
These findings suggest a potential class effect despite the
varied affinity of these compounds for different RTKs,
with VEGFR being notably a common target of all of
these TKIs, and have generated excitement in the field.
However, different end points were used to define ther-
apeutic activity in some of the trials, particularly the
older studies, which has complicated cross-​trial compar-
isons to evaluate which agents to prioritize for further
evaluation.
Investigators have started to consider how to com-
bine TKIs with other agents to augment their activity.
The toxicities associated with combining TKIs with
chemotherapy are a major concern. Preliminary results
with the combination of lenvatinib with ifosfamide
and etoposide demonstrated two dose-​limiting toxici-
ties (grade 4 thrombocytopenia and grade 3 bleeding
in one patient, and grade 2 oral dysaesthesia, grade 2
back pain and grade 1 muscle spasms in another) when
lenvatinib was used at the recommended phase II
single-​agent dose of 14 mg/m2 (ref.63). No efficacy data
were reported for this combination and this trial is ongo-
ing (NCT02432274; Supplementary Table 2). Building
on their initial promising experience with single-​agent
sorafenib59, Grignani et al.65 performed a phase II trial
of sorafenib in combination with everolimus in patients
with relapsed, unresectable high-​grade osteosarcoma.
The primary end point of PFS ≥50% at 6 months was
not met, although the 6-​month PFS was 45%, with
66% of patients requiring dose reductions and/or short
treatment interruptions owing to toxicities65.
Some important concerns exist regarding the combi-
nation of multiple potentially active therapies. First, defin-
ing an appropriate end point for the combination therapy
of a novel agent with a known active chemotherapy is a
major challenge. Whether the end point typically applied
for single agents with unknown activity (4-month DCR
of 40%) can be extrapolated to combinations of agents
with known activity is unclear. Second, the addition
of the TKI might affect the administration of effective
therapeutic agents. Owing to these challenges, ongo-
ing clinical trials are evaluating TKIs as maintenance
therapy after the completion of planned chemother-
apy, including the placebo-​controlled REGOSTA trial
of regorafenib in patients with newly diagnosed bone
sarcomas (NCT04055220; Supplementary Table 2).
Indeed, an emphasis in drug development for oste-
osarcoma has been placed on identifying active agents
with non-​overlapping toxicities that might facilitate the
combination with drugs used as standard treatments.
Restricting drug development to certain novel classes
of agents with non-​overlapping toxicities or at least to
agents that are unlikely to necessitate changes to the
dose and intensity of the existing treatment and that
also demonstrate single-​agent activity in patients with
relapsed osteosarcoma might be a more appropriate
strategy. The COG has been developing a randomized
trial testing the incorporation of cabozantinib into
the upfront treatment of patients with osteosarcoma.
Notably, the involvement of cooperative groups is
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crucial to ensuring the timely completion of such studies
given the rarity of the disease and the large number of
patients needed. This requirement further supports the
stringency in agent selection.
SRC is a non-​receptor tyrosine kinase involved in
multiple signalling pathways, including the mTOR and
MEK–ERK pathways, and is activated in the majority
of osteosarcomas66. Dasatinib, a kinase inhibitor with
activity against SRC and ABL1, demonstrated tumour
growth inhibition in two of six osteosarcoma PPTC
PDX models, although no objective responses were
observed67. Moreover, a trial of saracatinib, a highly
selective inhibitor of SRC and ABL, was closed to accrual
after futility analysis did not demonstrate a PFS or OS
benefit post-​metastasectomy in patients with recurrent
osteosarcoma localized to the lung68.
Similarly to SRC, PDGFR has been shown to be
overexpressed in osteosarcomas, which has been asso-
ciated with an unfavourable prognosis69. However, treat-
ment with imatinib, a TKI with activity against PDGFR,
does not result in growth inhibition in preclinical mod-
els of osteosarcoma and was not efficacious in clinical
trials involving patients with this disease69,70. This raises
the concern that detecting the target in the tumour
might not suffice as a predictive biomarker and that
other active pathways might negate target inhibition
through redundancy, compensatory changes and other
mechanisms of resistance. Such mechanisms are also
likely to narrow the spectrum of tyrosine kinases that
might confer true dependencies underlying the class
effect of multi-​target TKIs70. Nevertheless, selected TKIs
are probably active agents in osteosarcoma, supporting
the current approach of using small-​cohort phase II tri-
als with a high efficacy threshold to identify promising
novel therapies.
Targeting the cell cycle and DNA repair. Multiple subsets
of osteosarcomas are characterized by somatic altera-
tions affecting the cell cycle and/or DNA damage repair
(DDR) pathways (Fig. 2) and clinical trials are being
designed to evaluate precision medicine approaches
predicated on such aberrations. For example, TP53
aberrations compromise the G1 cell cycle checkpoint,
thus increasing the dependence of tumour cells on the
G2 checkpoint to maintain DNA integrity and com-
plete cell division. Therefore, agents that disrupt the G2
checkpoint, such as WEE1 inhibitors, might enhance
the activity of DNA-​damaging agents and induce the
mitotic lethality of TP53-​mutant osteosarcoma cells.
Monotherapy with the WEE1 inhibitor AZD1775 has
limited activity in preclinical models of osteosarcoma;
however, efficacy is observed in some of the models
when this agent is combined with the topoisomerase 1
inhibitor irinotecan71. Similarly, combining WEE1 inhib-
itors with gemcitabine and radiation results in enhanced
efficacy in PDX models of osteosarcoma72,73. These data
highlight a potential mechanism to exploit the TP53
aberrations that are prevalent in osteosarcomas.
CDK2, CDK4/6 and Aurora kinases (AURKA and/or
AURKB) have been identified as other potential molec-
ular targets involved in the cell cycle (Fig. 2). CDK4 has
been demonstrated to be overexpressed in osteosarcoma
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PDX models39, cell lines and patient samples74. The inhi-
bition of CDK4/6 with palbociclib results in growth
inhibition in cell lines expressing high levels of CDK4
in vitro74. In vivo, single-​agent palbociclib induces
moderate tumour regression in a cisplatin-​resistant
PDX model but greater tumour regressions in com-
bination with sorafenib (as compared to either agent
administered as monotherapy)75. AURKA/B have also
been demonstrated to be overexpressed in osteosar-
coma patient samples76. The inhibition of AURKA led
to a maintained complete response (CR) in one of six
PDX models77 (Fig. 2); the inhibition of AURKB inhib-
ited osteosarcoma cell growth in vitro and in vivo78.
Disappointingly, a phase II trial of the AURKA inhibitor
alisertib demonstrated no responses among 10 patients
with osteosarcoma79. AURKB inhibitors have not been
evaluated in clinical trials in patients with osteosarcoma.
Dinaciclib, an inhibitor of CDK2, has been demon-
strated to induce apoptosis in osteosarcoma cell lines
in vitro80; this agent induced stable disease in one of the
five PDX models tested81 (Fig. 2). Dinaciclib has not yet
been evaluated in clinical trials in the setting of osteo-
sarcoma. These cell cycle targets require further study
to evaluate their potential role in the future treatment
of osteosarcoma.
DDR pathways are important targets in osteosarcoma
given the larger numbers of somatic alterations observed
in samples from patients22. As noted above, the somatic
alterations are mostly chromosomal breaks and translo-
cations rather than point mutations, indicative of defects
in DDR mechanisms22,33. Currently, a clinical trial is eval-
uating the combination of the PARP inhibitor olaparib
with the ATR inhibitor ceralasertib in patients with
recurrent osteosarcoma (NCT04417062; Supplementary
Table 2) based on the rationale that the replication stress
induced by these agents will lead to mitotic lethality in
tumour cells that have already largely lost control of
cell cycle checkpoints (owing to prevalent alterations in
TP53 and RB1). Defects in DDR pathways similarly sug-
gest the possibility for clinical trials of monotherapy with
these agents in biomarker-​selected patient populations.
Alterations in the ATR pathway constitute one potential
biomarker for the sensitivity to treatments targeting the
DDR in osteosarcoma82; however, this strategy needs to
be validated in prospective clinical trials.
Suppressing metastasis. Metastasis, most commonly
to the lungs, is a key determinant of the lethality of
osteosarcoma3. Substantial research has therefore been
focused on the development of agents to disrupt the
metastatic process. Such efforts have largely been driven
by the ‘seed and soil’ theory of metastasis, which posits
that a favourable microenvironment is required to create
a niche for the osteosarcoma metastases to take root. In
particular, FAS signalling and macrophages have been
identified as key factors mediating the formation of
osteosarcoma metastases83,84. The role of macrophages
is discussed further in the following section of this
Review. With regard to FAS, gemcitabine and entinostat
can augment the expression of this apoptotic receptor
in osteosarcoma lung nodules and induce the regression
of these lesions in preclinical models85,86. These findings
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have laid the foundation for an ongoing clinical trial end point of OS, leading to the approval of this agent
evaluating aerosolized gemcitabine (NCT03093909; by the EMA but not the FDA5. Subsequent studies have
Supplementary Table 2). IL-6 and CXCL8 have also demonstrated that mifamurtide is well tolerated and is
been identified as signals mediating osteosarcoma associated with a 1-​year and 2-​year OS of 71.7% and
homing to the lungs and the combined inhibition of 45.9%, respectively, in patients with resectable recur-
IL-6 and CXCL8 signalling abrogates the development rent and/or metastatic disease101. These outcomes were
of lung metastases in several models of osteosarcoma87. observed in a non-​randomized trial but exceed those
Similarly, CXCL13 induces osteosarcoma cell migration expected for children with recurrent and/or metastatic
and invasion in vitro88. osteosarcoma with surgery, with or without chemother-
More recently, the epigenomic profiling of osteosar- apy, suggesting that this agent provides a therapeutic
coma metastases has revealed that changes in the activity benefit. Mifamurtide is currently available through com-
of enhancers associated with several genes are neces- passionate use programmes in the USA. The disparate
sary to permit metastatic dissemination89. Preclinical responses by regulatory agencies continues to challenge
data indicate that such changes drive the expression the more widespread use of mifamurtide for the treat-
of distinct subsets of genes within the first 24 hours of ment of osteosarcoma, although an ongoing trial might
metastatic colonization and later during metastatic out- clarify the potential benefit of this agent (NCT03643133,
growth, while other genes are constitutively activated89. Supplementary Table 2).
These findings suggest that phenotypic plasticity and Alternative approaches to augment immune
adaptation is required throughout various stages of the responses have been investigated. For example, in the
metastatic process. Notably, pharmacological inhibition phase III EURAMOS-1 trial, the addition of IFNα2b
of the epigenetic reader BET or functional inhibition of as a maintenance therapy was evaluated in patients
individual genes activated by the enhancers, such as with a good pathological response to preoperative
that encoding coagulation factor III/tissue factor (F3), MAP chemotherapy102; those with a poor pathologi-
suppressed lung metastasis in models of osteosarcoma89. cal response were randomly assigned to receive either
Evidence indicates that the lung microenvironment also standard postoperative MAP or MAP plus ifosfamide
needs to be altered in order to permit the invasion and and etoposide (MAP-​IE)6. Although EURAMOS-1 was
growth of osteosarcoma, which might provide avenues an achievement in that it was an international study and
for novel treatments90,91. the largest prospective trial ever performed in patients
with osteosarcoma, neither the addition of IFNα2b nor
Immunotherapy for osteosarcoma ifosfamide and etoposide improved EFS6,102.
An immune component of the treatment of osteosar- Novel strategies to specifically activate TAMs
coma has been envisaged since William Coley first are being investigated, including inhibition of the
observed tumour regressions following treatment with CD47–SIRPα signalling pathway. CD47 acts as an
bacterial toxins92. This perspective has been supported immune checkpoint by inhibiting the phagocytosis
by circumstantial evidence indicating that patients of non-​malignant cells by binding to SIRPα on mac-
who develop surgical site infections following primary rophages. Tumour cells can co-​opt this mechanism as
tumour resection have better outcomes93,94. Analyses of a means of immune evasion, and inhibitory anti-​CD47
the tumour microenvironment (TME) of osteosarco- monoclonal antibodies (mAbs) are under clinical devel-
mas consistently demonstrate an immune cell infiltrate opment as anticancer agents103. Few responses were
consisting of both macrophages and T cells95–98. The observed in an initial phase I trial of the first-​in-​class
presence of tumour-​infiltrating lymphocytes (TILs) anti-​CD47 mAb magrolimab in patients with various
has led to the hypothesis that ICIs could be effective in advanced-​stage cancers103; however, this agent was very
patients with this disease. In addition, osteosarcoma well tolerated, which might increase the feasibility of
was one of the first diseases in which the activation of using it as an adjunct therapy to improve the efficacy
tumour-​associated macrophages (TAMs) was pursued of other immunotherapies. With regard to osteosar-
as a treatment strategy. coma, CD47 is typically expressed at higher levels in
the tumour cells than in the surrounding tissue, and
Targeting macrophages. Early studies with muramyl antagonistic antibodies to CD47 have been shown to
tripeptide phosphatidylethanolamine (MTP-​PE; also increase macrophage-​mediated phagocytosis in vitro
known as mifamurtide) revealed that this agent can and to decrease the development of pulmonary metas-
activate blood monocytes to selectively kill tumour tases in PDX models104. Clinical trials are currently being
cells but not non-​malignant cells83,99. Mifamurtide can designed to evaluate whether inhibiting CD47 augments
activate the innate immune system via the pattern-​ the activity of other immunotherapies in patients with
recognition receptor NOD2, which is highly expressed in osteosarcoma. In one such proposed trial, an anti-​CD47
monocytes100. The initial preclinical and clinical experi- antibody will be combined with an anti-​GD2 antibody105.
ence with mifamurtide led to a phase III trial evaluating Lung macrophages have also been implicated as fac-
the addition of this agent to standard chemotherapy for tors that foster a stable niche for osteosarcoma metas-
patients with resectable non-​metastatic osteosarcoma, tases. Vascular cell adhesion molecule 1 (VCAM1) has
which was complicated by its factorial design involving been demonstrated to be expressed on tumour cells and
the parallel randomized testing of ifosfamide. Ultimately, is associated with an increased ability to metastasize106.
mifamurtide did not meet the primary EFS end point Indeed, VCAM1 expressed by osteosarcoma cells
but did demonstrate benefit according to the co-​primary can interact with the integrins expressed on lung

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macrophages, which promotes cell migration and the col-
onization of the pre-​metastatic niche88. Researchers are
currently developing antagonistic anti-​VCAM1 mAbs
to abrogate this interaction with the goal of reducing
pulmonary metastases107.
Harnessing the immune infiltrate. In addition to TAMs,
TILs also have an important role in the immune response
to osteosarcoma, with ~75% of tumours expressing cell-​
surface markers consistent with T cell and macrophage
infiltrates98. However, an increased abundance of TILs is
correlated with the expression of PD-​L1 and the related
protein HHLA2 in osteosarcomas, which are associated
with an unfavourable EFS95,96. By contrast, an increased
ratio of CD8+ cytotoxic T cells to FOXP3+ regulatory
T cells in the TME has been associated with an improved
prognosis108. Further stratification revealed that nearly
100% of patients with a CD8+ to FOXP3+ T cell ratio
greater than the median were alive beyond 16 years
compared with <50% for those with a ratio less than the
median108. Together, these data highlight the importance
of tumour cell evasion of the immune infiltrate in the
pathogenesis of osteosarcoma. Accordingly, numerous
approaches are being taken to alleviate the immunosup-
pressive microenvironment of tumours, in particular
with ICIs or adoptive cell therapies.
One approach to harnessing the immune system
that is currently under investigation in osteosarcoma
involves harvesting TILs from patient surgical speci-
mens. These TILs are then expanded ex vivo, infused
back into the patient and activated in vivo through the
post-​infusion administration of IL-2 (NCT03449108)109.
This approach is antigen agnostic, with the presumption
that these TILs, given their numbers and extrinsic acti-
vation, can overwhelm the immunosuppressive mecha-
nisms of osteosarcomas and induce tumour regression.
Similarly, the adoptive transfer of natural killer (NK)
cells is another non-​specific mechanism of harness-
ing the immune system. NK cells are part of the innate
system and can recognize malignant cells through a sys-
tem of activatory and inhibitory receptors; these cells
have demonstrated preclinical activity against osteo-
sarcoma both in vitro and in vivo110–112. Clinical trials
designed to evaluate the efficacy of adoptive NK cell
therapy in patients with osteosarcoma are under way
(NCT03420963 and NCT02100891; Supplementary
Table 2). Moreover, newer iterations of NK cell therapy
analogous to the chimeric antigen receptor (CAR) T cell
approaches that are used in the treatment of B cell malig-
nancies create the opportunity to target specific antigens
expressed by osteosarcoma cells113.
Members of the B7 family of immune-​checkpoint
proteins, including PD-​L1, HHLA2 and B7-​H3 (also
known as CD276), have been demonstrated to be
expressed on the surface of osteosarcoma cells as a mech-
anism of immune evasion95,96,114,115. This observation has
led to multiple clinical trials of ICIs targeting PD-1, the
receptor for PD-​L1, in patients with advanced-​stage
osteosarcoma, with somewhat disappointing results.
For example, in the phase II PEMBROSARC trial of
pembrolizumab plus metronomic cyclophosphamide,
1 (6.7%) of 15 evaluable patients had a partial response
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(PR) and 5 (33.3%) had stable disease; the 6-​month PFS
was 13.3%116. In the phase II SARC study of pembroliz­
umab in patients with advanced-​stage sarcomas, only
1 (4.5%) of 22 patients with osteosarcoma enrolled had
a PR, with the majority discontinuing therapy owing to
disease progression117. The COG has also conducted a
phase I/II trial of nivolumab in paediatric patients with
relapsed solid tumours including 13 patients with oste-
osarcoma, all of whom had rapid disease progression
during treatment with this agent118. These results might
reflect fundamental differences between the biology
of paediatric and adult malignancies, in particular in
terms of tumour mutational burden and potential neo-
antigen expression. Osteosarcoma has one of the high-
est tumour mutational burdens of any paediatric cancer
type119; however, unlike most malignancies that occur
in adults, these mutations manifest as chromosomal
rearrangements rather than as point mutations37,119.
Moreover, the majority (>70%) of genomic events
observed occur in non-​coding regions. Tumours with a
high predicted neoantigen expression also have high lev-
els of nonsense-​mediated decay factors, although such
tumours typically have higher immune scores37.
Despite these disappointing results, the biology of
immune checkpoints in osteosarcoma continues to drive
interest in this treatment approach. In mouse models
of this disease, PD-1 inhibition has been demonstrated
to decrease lung metastasis via the conversion of mac-
rophages from a pro-​tumour M2 phenotype into an
antitumour M1 phenotype120. These findings provide
a potential rationale for further clinical trials aim-
ing to improve the responses to ICIs (Supplementary
Table 2). Avelumab, an mAb targeting PD-​L1 rather than
PD-1, is being evaluated in a multicentre phase II trial
in patients with recurrent or progressive osteosarcoma
(NCT03006848). In an alternative approach, azacitidine
is being explored in patients with resectable relapsed
osteosarcoma with the goal of increasing neoanti-
gen expression and thereby increasing the efficacy of
nivolumab (NCT03628209)121. The COG is currently in
the planning stages of a trial using radiation as another
mechanism of increasing neoantigen expression as well
as inducing other immunological changes and augment-
ing responses to ICIs122. ICIs are also being combined
with the anti-​CD73 antibody oleclumab in a study
involving patients with advanced-​stage osteosarcoma
(NCT04668300; Supplementary Table 2). This approach
is predicated on the finding that tumours express-
ing CD73 accumulate extracellular adenosine, which
impairs antitumour T cell responses and thus constitutes
another mechanism of immune evasion123; therefore, the
hope is that the combined inhibition of PD-1 or PD-​L1
and CD73 will increase the T cell response to osteosar-
coma. B7-​H3 is also being explored as a therapeutic
target in osteosarcoma (as is discussed further below),
although using agents distinct from ICIs.
Exploiting the osteosarcoma surfaceome
A number of proteins are known to be enriched on the
surface of osteosarcoma cells (Fig. 4), and research to dis-
cover additional clinically applicable cell-​surface targets
is ongoing. Owing to the rarity of osteosarcoma, clinical
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ADC (samrotamab
ADC vedotin)
(glembatumumab Positive preclinical CAR T cells
vedotin) results (EGFR806-EGFRt) ADCs (e.g.
Negative phase II Clinical activity in Phase I trial omburtamab,
trial phase I trial ongoing m276-PBD,
ADC MGC018 and
(trastuzumab Bi-specific DS-7300a)
mAb deruxtecan) T cell engager Positive preclinical
(trastuzumab) Phase II trial (Hu3F8-BsAb) results (m276-PBD)
Negative ongoing Phase I/II trial
phase II trial ongoing

GD2 LRRC15
GPNMB EGFR
HER2 B7-H3
mAb CAR T cells
Negative (e.g. GD2CART, CAR T cells
CAR T cells phase II trial iC9-GD2-CAR-VZV-CTL and
(HER2-CD28 (B7H3-EGFRt-DHFR)
(dinutuximab) GD2-CAR.OX40.28.z.ICD9) Positive preclinical
T cells) Phase I/II trial Phase I trials ongoing
Phase I trial results
ongoing Phase I trial ongoing
ongoing (Hu3F8)

Fig. 4 | Targeting the surfaceome of osteosarcoma. A number of cell-​surface molecules are commonly overexpressed on
osteosarcoma cells. Accordingly, a number of antibody-​based and/or cell therapy approaches targeting these molecules
are being studied for the treatment of osteosarcoma. For each approach, the stage of investigation or the results of trials
reported thus far are summarized. ADC, antibody–drug conjugate; CAR, chimeric antigen receptor; mAb, monoclonal
antibody.

trials investigating strategies to target components of the
‘surfaceome’ of this disease have mostly used agents that
were developed for overlapping high-​priority targets in
other, more common malignancies.
GPNMB. GPNMB has physiological roles in osteo-
blast differentiation and osteoclast development and
has been demonstrated by immunohistochemistry to
be expressed in 92.5% of osteosarcoma samples. The
GPNMB-​targeted ADC glembatumumab vedotin, which
has the antimitotic microtubule inhibitor monomethyl
auristatin E (MMAE) as a payload, has demonstrated
in vitro and in vivo preclinical activity124,125. However, in
a phase II trial43, glembatumumab had limited activity
against relapsed and/or refractory osteosarcoma (Fig. 4).
Of the 22 patients enrolled, 1 patient had a PR (4.5%)
and 2 patients had stable disease (9.1%)43.
LRRC15. Leucine-​ r ich repeat-​ c ontaining protein
15 (LRRC15) is a member of the leucine-​rich repeat
superfamily of proteins, which have been implicated
in cell adhesion, invasion and immune responses,
among other functions. Most osteosarcomas (91.3%)
have some degree of staining for LRRC15 on immuno­
histochemistry126. Samrotamab vedotin (also known
as ABBV-085) is an ADC comprising an anti-​LRRC15
mAb conjugated to MMAE (Fig. 4). Preclinically, samro-
tamab vedotin resulted in the prolongation of EFS in five
of seven PPTC PDX models of osteosarcoma in compar-
ison with an isotype control ADC, with a maintained
CR observed in two models127. Responses correlated
with the tumoural level of LRRC15 expression128. Data
from the first-​in-​human phase I trial of samrotamab
vedotin in 78 patients with various malignancies
demonstrated PRs in 4 (14.8%) and stable disease in
8 (29.6%) of 27 patients with sarcoma, 10 of whom had
osteosarcoma129. The limited level of activity observed
with samrotamab vedotin as well as with glembatu-
mumab vedotin might reflect the intrinsic activity of
their MMAE payload: the PPTC results with microtu-
bule inhibitors in PDX models of osteosarcoma con-
sistently demonstrate mixed responses, with some PDX
models being highly resistant to these agents (Fig. 3).
Moreover, these responses, although better than those
observed with other agents (such as topoisomerase
inhibitors), have not been recapitulated clinically as best
demonstrated by the lack of responses to eribulin in a
phase II trial involving patients with relapsed metastatic
osteosarcoma44. Thus, reconstructing these ADCs with
a payload that has a greater efficacy in osteosarcoma,
such as with pyrrolobenzodiazepine (PBD) or other
DNA-​damaging agents, might increase their efficacy.
Nevertheless, consideration should also be given to the
importance of the target antigen.
HER2. HER2 is expressed in ~40% of osteosarcomas
and has been heavily explored as a therapeutic target in
this disease, although with contradictory results130–132.
In a trial with biomarker-​stratified treatment alloca-
tion, the addition of anti-​HER2 mAb trastuzumab to

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upfront MAP-​IE chemotherapy for patients with HER2-​
overexpressing metastatic osteosarcoma resulted in a
similar 30-​month EFS and OS to that observed with
MAP-​IE alone in those without HER2 overexpression
(32% versus 32% and 59% versus 50%, respectively)133.
Whether HER2 is prognostic in osteosarcoma has been
a controversial question; however, data derived from
233 patients enrolled on prospective clinical trials indi-
cate that HER2 overexpression is not prognostic in terms
of EFS or OS131. In contrast with trastuzumab, HER2-​
targeted CAR T cell therapy has some activity against
HER2+ recurrent and/or metastatic osteosarcoma, with
3 of 14 evaluable patients having stable disease for between
12 and 15 weeks before undergoing tumour resection, 1
of whom had 90% tumour necrosis; these patients all
remained in remission after 6, 12 and 16 months with no
further treatment134. Updated data from this trial indi-
cate that 1 patient has an ongoing CR for 32 months135.
In addition, another patient with osteosarcoma had a PR
lasting 9 months after receiving salvage chemotherapy
and a second dose of CAR T cells for initial progressive
disease134. Other agents with demonstrated activity even
in tumours with low levels of HER2 expression, such
as trastuzumab deruxtecan136, are also being explored
in patients with osteosarcoma (Fig. 4; Supplementary
Table 2). Trastuzumab deruxtecan, an ADC with a topo­
isomerase inhibitor payload, prolonged the EFS in five of
seven PDX models of osteosarcoma (as compared with
vehicle controls)137. This work laid the foundation for a
clinical trial of this agent in patients with osteosarcoma
by the COG (NCT04616560). A potential limitation of
HER2-​directed therapy relates to a lower prevalence and
more heterogeneous levels of HER2 expression in oste-
osarcomas as compared with the other target antigens
discussed in this section.
GD2. GD2 is a disialoganglioside demonstrated to
be stably expressed on the surface of osteosarcoma
cells138,139. In humans, the expression of this glycosphin-
golipid in non-​malignant tissues is highly restricted,
mostly to the cerebellum and peripheral nerves, raising
the possibility for relatively specific tumour targeting.
Dinutuximab (Ch14.18) is a chimeric mouse–human
mAb targeting GD2 that can induce antibody-​dependent
cellular phagocytosis, antibody-​dependent cellular cyto-
toxicity and/or complement-​dependent cytotoxicity140.
Accordingly, GM-​CSF is typically used in conjunction
with this agent to help stimulate the anticancer activity of
TAMs. In a phase II trial of dinutuximab plus GM-​CSF
in patients with completely resected recurrent pulmo-
nary osteosarcoma, 12-​month EFS was 30.7%, which
did not meet the efficacy end point141. A phase II trial
of humanized 3F8 (Hu3FB), another chimeric mAb
targeting GD2, plus GM-​CSF is currently ongoing in a
similar population of patients in second or later com-
plete remission (NCT02502786; Supplementary Table 2).
The rationale for using these agents in the maintenance
setting is drawn from the experience with approved anti-​
GD2 mAbs in neuroblastoma, another GD2-​expressing
paediatric tumour type, in which the greatest benefit is
achieved when they are used for maintenance therapy
following initial disease control. In addition, several
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GD2-​targeted CAR T cell products as well as a Hu3FB-​
based bispecific T cell-​engaging construct have been
developed and are currently being tested in early phase
clinical trials (Fig. 4; Supplementary Table 2).
B7-​H3. B7-​H3 overexpression has been detected by
immunohistochemistry in 91.8% of osteosarcoma
specimens114. Unlike other immune-​checkpoint proteins,
antagonistic mAbs are not being used to directly inhibit
B7-​H3 signalling and thereby activate an immune
response but rather B7-​H3 is being exploited for the tar-
geting of ADCs and CAR T cells towards osteosarcoma
cells (Fig. 4). This strategy at least partially reflects the fact
that the cellular function of B7-​H3 remains unclear142.
The targeting of B7-​H3 was first explored in preclini-
cal models of osteosarcoma and other cancers using the
mAb omburtamab (8H9) conjugated to a Pseudomonas
exotoxin143. Owing to systemic toxicities observed in
these studies, intraperitoneal (NCT04022213) or intrathe­
cal (NCT03275402 and NCT00089245) administration
of this agent conjugated to the radioisotope iodine-131 is
being evaluated in patients with other paediatric malig-
nancies. B7-​H3-​targeted ADCs are also under devel-
opment, including m276-​PBD144,145. This agent carries
a DNA-​damaging agent PBD payload and induced
CRs in two of five PDX models of osteosarcoma144,145.
Another ADC targeting B7-​H3, MGC018, has a DNA-​
alkylating payload (duocarmycin) and has demonstrated
potent antitumour activity in mouse models of various
adult carcinomas146. This agent is currently undergoing
evaluation in osteosarcoma PDX models by the PPTC.
Preliminary data from the first-​in-​human phase I/II trial
of MGC018 (NCT03729596), which did not include
patients with osteosarcoma, has revealed a manageable
safety profile and evidence of activity in 4 of 20 patients
enrolled thus far147. DS-7300a, another B7-​H3-​targeted
ADC, has a topoisomerase inhibitor payload and is cur-
rently being evaluated in a phase I/II trial in adults with
advanced-​stage solid tumours (NCT04145622).
With regard to cell therapy strategies, B7-​H3-​targeted
CAR T cells have demonstrated potent antitumour
activity in one osteosarcoma xenograft model148. A
clinical trial of this CAR T cell product with a trun-
cated EGFR-​based safety switch is currently open to
paediatric patients with osteosarcoma (NCT04483778;
Supplementary Table 2). Selection of patients based on
the cell-​surface expression of B7-​H3 is not required
given the prevalence of this antigen in paediatric cancers.
EGFR. Anti-​EGFR CAR T cells are being investigated in
paediatric and young adult patients with osteosarcoma
(NCT03618381; Supplementary Table 2). The major-
ity of osteosarcoma cell lines (10 patient-​derived and
4 standard commercially available cell lines) express low
levels of EGFR149. Moreover, the EGFR TKI gefitinib
has activity against cell lines with high expression
of EGFR only under conditions of serum starvation150.
EGFR has been found to be amplified in 28% of human
osteosarcomas151. A trial of EGFR-​targeted CAR T cells
in patients with glioblastoma demonstrated trafficking
of the cells to the tumour bed following intravenous
administration, with limited systemic toxicity; however,
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 Reviews

analyses of post-​infusion resected specimens revealed
the downregulation of cell-​surface EGFR expression and
a more immunosuppressive TME152.
CAR T cell versus ADCs. CAR T cells and ADCs have
a similar specificity for the same target antigen; how-
ever, rather than requiring the antigen–antibody com-
plex to deliver the payload, the CAR T cell approach
exploits the intrinsic cytotoxicity machinery of T cells
to eliminate tumour cells. CAR T cells have the addi-
tional benefit that they can persist in vivo for prolonged
periods of time, leading to continued eradication of cells
expressing a particular antigen153. CAR T cells might
also circumvent the challenge with ADCs relating to
the requirement for a payload that is active against oste-
osarcoma. Nevertheless, CAR T cell therapy is associ-
ated with concerns, including limited tumour homing,
cytokine-​related toxicities, on-​target off-​tumour effects
and cellular exhaustion. NK cells incorporating the CAR
technology (CAR NK cells) are being explored as an
alternative to overcome these potential drawbacks. For
example, CAR NK cells do not seem to carry the same
risk of inducing cytokine storms and are able to survive
after killing multiple target cells, which in T cells can
lead to exhaustion or activation-​induced cell death113.
Conclusions
Over the past decade, the fruits of years of research using
tissue banks, the development of preclinical models and
genomic characterization have led to unprecedented
advances in our understanding of osteosarcoma. The
molecular characterization of osteosarcoma PDXs and
patient samples have raised the possibility of designing
biomarker-​stratified clinical trials. Innovative approaches
to drug development using small phase II trials with a
high bar for activity have provided the foundation for
the rapid clinical evaluation of a multitude of novel
therapies. Furthermore, we recognize that new molec-
ular techniques, such as single-​cell RNA sequencing to
characterize tumour heterogeneity as well as the immune
infiltrate and CRISPR or Sleeping Beauty screens for
novel therapeutic targets, will continue to enrich our
understanding of osteosarcoma as they are applied to
this disease in the very near future, which will facilitate
the prioritization of treatments that are most likely to
be effective154,155. The use of techniques such as multi-
plexed ion beam imaging enables staining for 40 markers
simultaneously in paraffin-​embedded tissue specimens,
thus permitting rapid screening of patients for possible
enrolment in biomarker-​based trials156. Such a strategy is
essential in conducting clinical trials of targeted therapies
in a very heterogeneous patient population.
The role of cell-​surface antigen expression in oste-
osarcoma is an area that is clearly under active inves-
tigation with hopes of improving the treatment of the
disease. Several targets are currently being evaluated
(Supplementary Table 2) with more coming down the
pipeline. As discussed, these nascent strategies in oste-
osarcoma face several hurdles. First, the antigen needs
to be of sufficient functional importance to avoid the
treatment-​related clonal selection of tumour cells with
low or no expression of the target, particularly consider-
ing the known intratumoural heterogeneity of osteosar-
coma. Second, the optimal method of targeting, whether
with an mAb (typically requiring sufficient immune
activation), an ADC (requiring an active payload) or a
cell therapy product (avoiding exhaustion), remains to
be determined for most if not all antigens and requires
further clinical studies.
Ultimately, novel agents will probably need to be
combined in order to overcome the intrinsic treatment
resistance observed in the osteosarcoma clinical trials
of the past. As noted in the example of lenvatinib63, the
high doses of cytotoxic chemotherapy currently used in
patients with osteosarcoma leave limited space for addi-
tional agents without prohibitive toxicities. Reducing
doses or the number of cycles might compromise the
outcomes achieved thus far. TKIs still remain the next
likely choice of agents to be incorporated with upfront
chemotherapy, whether as maintenance therapy, as
advocated by some who are concerned by their toxicities,
or by developing novel strategies to safely integrate them
into the current chemotherapy regimens.
The interest in immune-​based therapies is height-
ened by their different toxicity profiles, which do not
necessarily overlap with known toxicities of cytotoxic
chemotherapy. This scenario might be favourable for
integrating immunotherapies into current treatment
regimens if they are first found to be efficacious in
clinical trials, similar to the strategy currently being
applied in clinical trials focused on paediatric haemato-
logical malignancies (for example, NCT02166463 and
NCT03914625).
Immune-​based treatments might also have a role in
maintenance therapy, similar to that of anti-​GD2 mAbs
in patients with neuroblastoma. Indeed, these immuno­
therapies are likely to be most effective in the setting
of minimal residual disease. Most patients with osteo-
sarcoma who have disease recurrence have lung-​only
metastasis, which probably occurs owing to the pres-
ence of micrometastases at the time of diagnosis. The
treatments outlined herein provide potential avenues
to eradicate these occult micrometastases before they
develop into overt metastatic disease. With all of the
new biological discoveries, technologies, agents and
approaches, osteosarcoma seems to be a disease on the
verge of marked advancement in patient outcomes after
more than four decades of stagnation.
Published online xx xx xxxx

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Acknowledgements
The work of R.G. is supported by The University of Texas MD
Anderson Cancer Center as the H. Grant Taylor, M.D., W.W.
Sutow, M.D. and Margaret P. Sullivan, M.D. Distinguished
Chair in Pediatrics. J.G. and R.G. both acknowledge support
from The Foster Foundation, Swim Across America,
the Osteosarcoma Institute, the QuadW Foundation and the
Barbara Epstein Foundation.
Author contributions
Both authors contributed to all aspects of the preparation of
this manuscript.
Competing interests
The authors declare no competing interests.
Peer review information
Nature Reviews Clinical Oncology thanks Bruno Fuchs,
Dominique Heymann, Massimo Serra and the other, anony-
mous, reviewer(s) for their contribution to the peer review of
this work.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
Supplementary information
The online version contains supplementary material available
at https://doi.org/10.1038/s41571-021-00519-8.
Related links
Bone Cancer Research Trust: https://www.bcrt.org.uk/
Children’s Oncology Group (COG): https://
childrensoncologygroup.org/index.php/
childrens-​oncology-​group
Count Me in: https://www.broadinstitute.org/count-me-in
euroBoNeT: https://cordis.europa.eu/project/id/18814
iTCC P4: https://www.itccp4.eu
National Cancer institute (NCi) Therapeutically Applicable
Research to Generate effective Treatments (TARGeT)
Osteosarcoma project: https://ocg.cancer.gov/programs/
target/projects/osteosarcoma
NCi Paediatric Preclinical Testing Consortium: http://www.
ncipptc.org/
Osteosarcoma Project: http://www.osproject.org
Quadw Foundation: http://www.quadw.org/
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