Immunotherapy for SMARCB1-Deficient Sarcomas: Current Evidence and Future Developments

Abstract

Mutations in subunits of the SWItch Sucrose Non-Fermentable (SWI/SNF) complex occur in 20% of all human tumors. Among these, the core subunit SMARCB1 is the most frequently mutated, and SMARCB1 loss represents a founder driver event in several malignancies, such as malignant rhabdoid tumors (MRT), epithelioid sarcoma, poorly differentiated chordoma, and renal medullary carcinoma (RMC). Intriguingly, SMARCB1-deficient pediatric MRT and RMC have recently been reported to be immunogenic, despite their very simple genome and low tumor mutational burden. Responses to immune checkpoint inhibitors have further been reported in some SMARCB1-deficient diseases. Here, we will review the preclinical data and clinical data that suggest that immunotherapy, including immune checkpoint inhibitors, may represent a promising therapeutic strategy for SMARCB1-defective tumors. We notably discuss the heterogeneity that exists among the spectrum of malignancies driven by SMARCB1-loss, and highlight challenges that are at stake for developing a personalized immunotherapy for these tumors, notably using molecular profiling of the tumor and of its microenvironment.

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Citation: Ngo, C.; Postel-Vinay, S.
Immunotherapy for
SMARCB1-Deficient Sarcomas:
Current Evidence and Future
Developments. Biomedicines 2022,10,
650. https://doi.org/10.3390/
biomedicines10030650
Academic Editor: Francesca Lovat
Received: 12 February 2022
Accepted: 10 March 2022
Published: 11 March 2022
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biomedicines
Review
Immunotherapy for SMARCB1-Deficient Sarcomas:
Current Evidence and Future Developments
Carine Ngo 1,2 and Sophie Postel-Vinay 1,3 ,*
1
ATIP-Avenir Group, Inserm Unit U981, Gustave Roussy, 94800 Villejuif, France; carine.ngo@gustaveroussy.fr
2Department of Pathology, Gustave Roussy, 94800 Villejuif, France
3Drug Development Department, DITEP, Gustave Roussy, 94800 Villejuif, France
*Correspondence: sophie.postel-vinay@gustaveroussy.fr
Abstract:
Mutations in subunits of the SWItch Sucrose Non-Fermentable (SWI/SNF) complex occur
in 20% of all human tumors. Among these, the core subunit SMARCB1 is the most frequently
mutated, and SMARCB1 loss represents a founder driver event in several malignancies, such as
malignant rhabdoid tumors (MRT), epithelioid sarcoma, poorly differentiated chordoma, and renal
medullary carcinoma (RMC). Intriguingly, SMARCB1-deficient pediatric MRT and RMC have recently
been reported to be immunogenic, despite their very simple genome and low tumor mutational
burden. Responses to immune checkpoint inhibitors have further been reported in some SMARCB1-
deficient diseases. Here, we will review the preclinical data and clinical data that suggest that
immunotherapy, including immune checkpoint inhibitors, may represent a promising therapeutic
strategy for SMARCB1-defective tumors. We notably discuss the heterogeneity that exists among the
spectrum of malignancies driven by SMARCB1-loss, and highlight challenges that are at stake for
developing a personalized immunotherapy for these tumors, notably using molecular profiling of
the tumor and of its microenvironment.
Keywords:
SMARCB1; immunotherapy; immune checkpoint inhibitor; epithelioid sarcoma; rhabdoid
tumor; SWI/SNF
1. Introduction
Sarcomas represent a very heterogeneous group of rare soft tissue and bone cancers,
which comprise more than 100 subtypes, and arise in all age groups, making it particularly
difficult to treat. Sarcomas have traditionally been classified according to their histopatho-
logical aspect, together with some specific immunohistochemical markers [
1
]. The advent
of next generation sequencing techniques (notably whole exome and RNA sequencing),
has allowed to build a new molecular classification of sarcomas, notably based on mu-
tations, copy number alterations, and gene fusions [
2
]. More recently, other classifiers
have emerged, notably based on DNA methylation profiles [
3
], or on digital pathology
associated with artificial intelligence/deep learning analyses, which all represents valuable
additional tools for sarcoma classification [4].
Beyond genetic abnormalities, several sarcoma subtypes are driven, at least in part,
by epigenetic dysregulation. Notably, alterations in subunits of the mammalian SWItch
Sucrose Non-Fermentable (mSWI/SNF) chromatin remodeling complex have been found
in 20% of human cancers and are frequent in certain sarcoma subtypes [
5
]. SWI/SNF
plays essential roles in chromatin organization, genome maintenance, and gene regula-
tion [6]. Specifically, the core subunit SMARCB1 (INI1), is the most frequently inactivated
subunit in mesenchymal neoplasms [
7
], with SMARCB1 deficiency being characteris-
tic of malignant rhabdoid tumors (MRT), epithelioid sarcoma (ES), and most of poorly
differentiated chordoma.
Despite the tremendous heterogeneity of sarcomas and the impressive progresses
made in understanding the disease biology thanks to the molecular classification, treatment
Biomedicines 2022,10, 650. https://doi.org/10.3390/biomedicines10030650 https://www.mdpi.com/journal/biomedicines

Biomedicines 2022,10, 650 2 of 17
still most often relies on “one size fits all” therapeutic strategy, based on poly-chemotherapy,
surgery, and radiotherapy, which brings little benefit at the cost of high toxicity. Over the
past ten years, cancer immunotherapy particularly with immune checkpoint inhibitors
(ICI) targeting the Programmed Death-1 (PD-1)/ligand-1 (PD-L1) axis, has revolutionized
the outcome of several aggressive cancers, such as metastatic melanoma, non-small-cell
lung cancer (NSCLC) or renal cell carcinoma [
8
]. In sarcoma, development of immunother-
apy has encountered challenges due to the rarity and high heterogeneity of the disease.
Nonetheless, recent scientific advances and clinical results have enabled the identification
of sarcoma subgroups that may most benefit from ICI, together with potential predic-
tive biomarkers of efficacy. Notably, mSWI/SNF defects emerged recently as a putative
promising predictive biomarker of sensitivity to ICI [9,10].
This review will discuss the current evidence for using immunotherapy in SMARCB1-
deficient sarcoma.
2. SMARCB1
SMARCB1 (aka SNF5, INI1 and BAF47), is one of SWI/SNF core subunits. mSWI/SNF
remodeling complexes, also known as BRG1/BRM-associated factor (BAF) complexes, exist
in three forms: the canonical BAF (cBAF), Poly-Bromo BAF (PBAF), and non-canonical BAF
(ncBAF), which differ in subunit composition and patterns of genomic targeting [
6
]. Each
complex is composed of three or five core subunits (SMARCC1, SMARCC2, SMARCD1-3,
SMARCB1, and SMARCE1 for cBAF and PBAF; SMARCC1, SMARCC2 and SMARCD1-3
for ncBAF), one ATPase subunit (SMARCA2 or SMARCA4 for cBAF and PBAF), some
complex-specific subunits, and several additional regulatory subunits [
6
,
11
]. The mSWI/SNF
complexes are involved in multiple important cellular processes, including cell differ-
entiation and cell proliferation [
12
], or transcription by facilitating the binding of the
transcription complex machinery and specific transcription factors to target genes [6].
2.1. SMARCB1 Structure and Functions
The SMARCB1 gene, located at 22q11.23, encodes a 47 kDa protein of four func-
tional domains with frequent loss of function mutations in cancer [
13
] (Figure 1). Interest-
ingly, SMARCB1 was first identified as a binding partner of the human immunodeficiency
virus-type 1 (HIV-1) integrase (to which it binds through its two highly conserved im-
perfect repeat domains, Rpt1 and Rpt2), and was, thereafter, named integrase interactor
1,
INI1 [14,15]
. Through its Rpt1 domain, SMARCB1 further interacts with the SWIRM
domain of SMARCC1 to form the BAF and PBAF forms of mammalian SWI/SNF com-
plexes [
16
]. Very recently, this N-terminal region, which is notably mutated in rhabdoid
tumors, was further found to undergo a coil-to-helix transition upon binding of SMARCC1,
revealing a unique binding interface [
17
]. SMARCB1 also contains a N-terminal winged
helix DNA-binding domain, which is regularly mutated in schwannomatosis [
18
]. The
highly conserved putative coiled-coil C-terminal
α
helix domain (CTD)—also frequently
altered in cancer—was found to mediate chromatin remodeling through interaction with
the acidic patch of the nucleosome [19].
SMARCB1 plays a key role in maintaining SWI/SNF integrity [
11
], which is required
for its targeting to regulatory regions where it opposes Polycomb-mediated repression
at bivalent promotors [
20
,
21
]. SMARCB1 also commonly acts as a tumor suppressor by
transcriptionally regulating the cell cycle, proliferation, and differentiation. For example,
SMARCB1 has been shown to regulate the activation of CyclinD1/CDK4 signaling, to
repress RB target genes, and to participate to the regulation of c-MYC-associated transcrip-
tional programs [22].

Biomedicines 2022,10, 650 3 of 17
Biomedicines 2022, 10, x FOR PEER REVIEW 3 of 18
Figure 1. Schematic representation of SMARCB1 functional domains and summary of pathogenic
somatic mutations (COSMIC). The SMARCB1 protein contains four functional domains: a winged
helix domain DNA-binding domain, DBD (aa10-110), two highly conserved imperfect repeat do-
mains, Rpt1 (aa186-248) and Rpt2 (aa259-319) and the highly conserved putative coiled-coil C-ter-
minal α helix domain, CTD (aa335-375). Synonymous mutations have been excluded. Number of
the most frequent pathogenic somatic mutations found in COSMIC are indicated.
SMARCB1 plays a key role in maintaining SWI/SNF integrity [11], which is required
for its targeting to regulatory regions where it opposes Polycomb-mediated repression at
bivalent promotors [20,21]. SMARCB1 also commonly acts as a tumor suppressor by tran-
scriptionally regulating the cell cycle, proliferation, and differentiation. For example,
SMARCB1 has been shown to regulate the activation of CyclinD1/CDK4 signaling, to re-
press RB target genes, and to participate to the regulation of c-MYC-associated transcrip-
tional programs [22].
2.2. SMARCB1 Alteration in Cancers
The mSWI/SNF complexes were first linked to cancer in 1998 after O. Delattre and
colleagues identified SMARCB1 biallelic inactivation as a driver of MRT [23]. Subse-
quently, complete loss of SMARCB1 expression has been found in a variety of tumors
(Table 1), including malignancies of the central nervous system, soft tissue, kidney, si-
nonasal and gastrointestinal tract [24]. Here, we will focus on SMARCB1-deficient sarcomas.
Table 1. SMARCB1-deficient malignant neoplasms.
SMARCB1-Deficient Mesenchymal
Malignant Tumors
SMARCB1-Deficient Non-Mesenchymal
Malignant Tumors
Extrarenal malignant rhabdoid tumor Atypical teratoid rhabdoid tumor
Epithelioid sarcoma Cribriform neuroepithelial tumor
Poorly differentiated chordoma Renal medullary carcinoma
Epithelioid MPNST 1 SMARCB1-deficient sinonasal carcinoma
Myoepithelial carcinoma SMARCB1-deficient carcinoma of the GI tract
Myxoid extraskeletal chondrosarcoma
1 malignant peripheral nerve sheath tumor.
Malignant rhabdoid tumor (MRT) are a rare, highly aggressive tumor that predomi-
nantly affects infants and young children below 3 years of age. They may affect the kidney
(Rhabdoid Tumor of the Kidney, RTK), central nervous system (Atypical Teratoid
Rhabdoid Tumor, ATRT) and extrarenal sites (Extra-Renal Rhabdoid Tumor, ERRT). They
present a remarkably simple genome and are uniquely characterized by a biallelic deletion
of SMARCB1 in more than 95% of cases [25–27]. In mice, whereas homozygous
Figure 1.
Schematic representation of SMARCB1 functional domains and summary of pathogenic
somatic mutations (COSMIC). The SMARCB1 protein contains four functional domains: a winged
helix domain DNA-binding domain, DBD (aa10-110), two highly conserved imperfect repeat domains,
Rpt1 (aa186-248) and Rpt2 (aa259-319) and the highly conserved putative coiled-coil C-terminal
α
helix domain, CTD (aa335-375). Synonymous mutations have been excluded. Number of the most
frequent pathogenic somatic mutations found in COSMIC are indicated.
2.2. SMARCB1 Alteration in Cancers
The mSWI/SNF complexes were first linked to cancer in 1998 after O. Delattre and
colleagues identified SMARCB1 biallelic inactivation as a driver of MRT [
23
]. Subsequently,
complete loss of SMARCB1 expression has been found in a variety of tumors (Table 1),
including malignancies of the central nervous system, soft tissue, kidney, sinonasal and
gastrointestinal tract [24]. Here, we will focus on SMARCB1-deficient sarcomas.
Table 1. SMARCB1-deficient malignant neoplasms.
SMARCB1-Deficient Mesenchymal
Malignant Tumors
SMARCB1-Deficient Non-Mesenchymal
Malignant Tumors
Extrarenal malignant rhabdoid tumor Atypical teratoid rhabdoid tumor
Epithelioid sarcoma Cribriform neuroepithelial tumor
Poorly differentiated chordoma Renal medullary carcinoma
Epithelioid MPNST 1SMARCB1-deficient sinonasal carcinoma
Myoepithelial carcinoma SMARCB1-deficient carcinoma of the GI tract
Myxoid extraskeletal chondrosarcoma
1malignant peripheral nerve sheath tumor.
Malignant rhabdoid tumor (MRT) are a rare, highly aggressive tumor that predomi-
nantly affects infants and young children below 3 years of age. They may affect the kidney
(Rhabdoid Tumor of the Kidney, RTK), central nervous system (Atypical Teratoid Rhabdoid
Tumor, ATRT) and extrarenal sites (Extra-Renal Rhabdoid Tumor, ERRT). They present
a remarkably simple genome and are uniquely characterized by a biallelic deletion of
SMARCB1 in more than 95% of cases [
25
–
27
]. In mice, whereas homozygous inactivation
leads to early embryonic lethality, heterozygous loss promotes the development of undiffer-
entiated or poorly differentiated sarcomas consistent with MRT observed in infants [
28
–
30
],
highlighting the central role of SMARCB1 in this disease.
Epithelioid sarcoma (ES) affects patients over a wide range of ages [
1
]. It is classically
classified in two clinicopathological subtypes: (i) the distal (or classical) type, which pre-
dominantly arises from the superficial distal sites and is most prevalent in adolescents
and young adults, is characterized by nodules of epithelioid to spindle cells with central
necrosis, surrounded by chronic inflammatory infiltrate; and (ii) the proximal type which
arises in deep central truncal sites in older patients, are characterized by sheet-like growth
of large, sometimes pleomorphic epithelioid tumor cells, with a poorer prognosis, and a
tendency to local recurrence and rapid metastasis. Rhabdoid cells may be seen in both
histological subtypes and some ES may demonstrate hybrid histological features, high-

Biomedicines 2022,10, 650 4 of 17
lighting the limitation of such classification. Median survival is ~52 weeks in patients with
metastasis [
31
]. Complete loss of SMARCB1 expression is found in more than 90% of both
subtypes [
32
]. Despite having many features overlapping those of ERRT, there are funda-
mental differences in clinical behavior, genomics, and presumably oncogenic mechanisms
between these tumor types. First, the mechanisms leading to SMARCB1 inactivation are
more diverse in ES than in MRT, and are still not fully understood. Genetic inactivation
of SMARCB1 have been reported to occur in 10% to 83% of cases [
33
–
36
], mostly in the
form of homozygous deletions and less frequently monoallelic deletion or more rarely
as nonsense, frame, or deleterious point mutations [
34
,
35
,
37
]. Epigenetic mechanisms,
notably following SMARCB1 mRNA downregulation by microRNAs [
38
–
40
], have also
been described. Unlike MRT, ES presents a moderate to high tumor mutation burden [
36
].
Further, the sometimes co-occurring loss of CDKN2A suggests that oncogenic mechanisms
are different than those reported in MRT—where SMARCB1 loss leads to cell cycle deregu-
lation through CDKN2A [
36
,
41
]. Altogether, this suggests that, contrary to MRT, additional
genomic abnormalities cooperate with SMARCB1 loss to drive ES. Treatment of localized ES
relies on surgery, most often subsequent to neoadjuvant chemotherapy and/or followed by
adjuvant radiation therapy [
42
]. Unfortunately, relapse or local recurrence frequently occur,
especially for proximal ES, with nodal or metastatic involvement. In 2020, tazemetostat
(Tazverik
®
, Epizyme) was the first ever epigenetic therapy to be approved in solid tumors.
Tazemetostat was registered for the treatment of advanced SMARCB1-defective ES. Still,
only 15% of patients respond to EZH2 inhibitors [
43
] and resistance rapidly develops,
urgently calling for complementary therapeutic approaches.
Poorly differentiated chordoma is an extremely rare tumor which mostly arises in the
skull base in young children, and more rarely in the sacrococcygeal area. It is characterized
by nests of epithelioid cells with notochordal differentiation and focal rhabdoid morphology,
and by the loss of SMARCB1 expression virtually in all cases [
44
]. Cytogenetic studies
have identified frequent homozygous deletions of SMARCB1 [
45
–
48
]. Poorly differentiated
chordoma is associated with a worse prognosis than that of conventional chordoma [
44
,
49
].
Treatment consists of a combination of surgery, radiation therapy, and chemotherapy.
Epithelioid malignant peripheral nerve sheath tumor, accounting for less than 5% of malig-
nant peripheral nerve sheath tumor (MPNST), differs from conventional MPNST by the strong
and diffuse expression of S-100 protein and SOX10, a rare association with type 1 neurofibro-
matosis, occasional origin in a schwannoma, and loss of expression of SMARCB1 following
homozygous deletion, nonsense, frameshift, or splice site mutations in up to 75% of cases [
50
,
51
].
Other SMARCB1-negative sarcomas. Loss of SMARCB1 expression has also been
observed in up to 40% of myoepithelial carcinoma, notably following homozygous dele-
tion [
52
,
53
], and in 17% of extra-skeletal myxoid chondrosarcoma [
54
]. Finally, synovial
sarcoma (SS) also shows reduced SMARCB1 function, following its eviction from the
SWI/SNF complex subsequent to the incorporation of the hallmark SS18–SSX oncopro-
tein [
55
]. Because this causes an indirect SMARCB1 functional defect rather than a direct
loss of the protein expression, we will not discuss SS here.
3. SMARCB1 Deficiency and Anti-Tumor Immunity
The immune system plays a key role in controlling tumor development and metastasis.
The tumor immune microenvironment (TME) is traditionally classified into three pheno-
types: immune-inflamed, immune-excluded, and immune-desert [
56
]. Immune checkpoint
inhibitors (ICI), notably those targeting the PD-1/-L1 or CTLA-4/CD28 checkpoints, have
revolutionized the prognosis of several aggressive diseases [
57
]. ICI block signals that
inhibit CD8+ T-cell activation, thereby reinvigorating in situ anti-tumor responses. The
immune-inflamed profile, characterized by CD8+ and CD4+ tumor-infiltrating lymphocytes
(TILs), and PD-L1 expression on immune and tumor cells, is classically associated with bet-
ter response to ICI [
58
,
59
]. Here, we will present the current knowledge on the TME-related
factors and tumor-related factors, which support a potential role for immunotherapy in
SMARCB1-defective tumors, with a focus on ICI.

Biomedicines 2022,10, 650 5 of 17
3.1. SMARCB1 Deficiency and Tumor Cell Immunogenicity
Several tumor-related factors have been shown to influence response to immune
therapies, including the tumor mutational burden (TMB) and neoantigen load [
60
], or the
mutation in genes that influence tumor ability to escape immune surveillance [
56
] (e.g.,
MYC or RAS [
61
,
62
]) or respond to interferon-gamma (IFN
γ
) signaling [
63
]. Interestingly,
recent data have reported a link between deficiencies of certain mSWI/SNF subunits and
increased mutational load or enhanced interferon response [6,64,65].
SMARCB1 modulates tumor cell immunogenicity through various mechanisms. Using
immunocompetent mouse models and human tumors, Leruste and colleagues showed
that, despite their very simple genome and extremely low mutational burden, MRT are
immunogenic [
66
]. Mechanistically, SMARCB1 deficiency favored the de-repression of
multiple endogenous retroviral elements (ERVs), leading to cytosolic double-stranded RNA
(dsRNA) accumulation which activates cytoplasmic sensors, such as TLR3 and MDA5,
and subsequent cell-autonomous IFN-
α
and IFN -
λ
responses (Figure 2). Interestingly,
recent studies have also reported that de-repressed ERVs could generate tumor specific
antigens presented at the tumor cell surface by MHC-I molecules and recognized by
specific T-cells [
67
,
68
]. An increased anti-tumor innate immune response driven by ERVs
de-repression has recently been observed in multiple tumor types [
69
,
70
], and correlated to
responsiveness to anti-PD-L1 therapy [71].
Biomedicines 2022, 10, x FOR PEER REVIEW 6 of 18
Figure 2. Interplay between SMARCB1 deficiency and immune modulation in SMARCB1-deficient
MRT and RMC. Bottom right part of the tumor cell: in MRT, ERVs de-repression contributes to the
accumulation of the cytosolic double-stranded RNA (dsRNA) which are recognized by the Toll-like
receptor (TLR) 3 and MDA5 sensors. MDA5 binds to MAVS resulting in a signaling cascade which
promotes the phosphorylation and nuclear translocation of IRF3, and subsequent induction of type
I/III interferon-stimulated genes (ISG). This overall results in cytokine production which favors the
recruitment of TILs. Aberrantly expressed ERV may also contribute to the development of an adap-
tive immune response through the production of tumor associated neoantigens (TAA). Upper left
part of the tumor cell: in RMC and within a context of MYC-induced replication stress, dsDNA is
released in the cytoplasm, which activates the cGAS/STING DNA-sensing pathway. The DNA sen-
sor cGAS binds to dsDNA, which triggers the formation of cyclic G-AMP (cGAMP), and subse-
quently activates STING. cGAMP-bound STING recruits TBK1 and phosphotylates IRF3, which
translocates to the nucleus where it triggers the expression of ISG.
3.2. SMARCB1-Deficient Tumors’ Immune Microenvironment
3.2.1. Tumor-Infiltrating Lymphocytes (TILs)
The tumor immune microenvironment of pediatric cancers has traditionally been an-
ticipated to be poorly infiltrated or immune-excluded [74], notably as a consequence of
their simple genome and low TMB. Surprisingly, despite sharing the same unique ge-
nomic alteration, rhabdoid tumors are molecularly heterogeneous [75] at the epigenomic
and transcriptomic level.
By studying the DNA methylation, Chun and colleagues identified five MRT sub-
groups: (1) ATRT-MYC, (2) ATRT-TYR, (3) RTK, (4) ERRT, and (5) ATRT-SHH [72].
Groups 1, 3, and 4 presented a high expression of immune-related genes, notably those
involved in antigen processing, antigen presentation, T-cell activation and homing, and
innate immune response. This correlated with increased infiltration of CD8+ cytotoxic T-
cells by immunohistochemistry and enriched immune-related gene signatures in RNA-
Seq, alongside with increased infiltration of PD-L1+ CD68+ myeloid cells [72]. Similarly,
Leruste and colleagues found that the ECRT and ATRT-MYC subgroups were highly in-
filtrated by CD8+ T-cells. Using single cell RNA and T-cell receptor sequencing, they iden-
tified clonally expanded resident memory CD8+ T-cells, suggesting the presence of a local
tumor-specific anti-tumor immune response [66]. In mouse syngeneic models, MRT were
highly infiltrated in PD-1+ TILs and were highly sensitive to anti-PD-1 therapy (75% of
complete response and survival benefit).
Figure 2.
Interplay between SMARCB1 deficiency and immune modulation in SMARCB1-deficient
MRT and RMC. Bottom right part of the tumor cell: in MRT, ERVs de-repression contributes to the
accumulation of the cytosolic double-stranded RNA (dsRNA) which are recognized by the Toll-like
receptor (TLR) 3 and MDA5 sensors. MDA5 binds to MAVS resulting in a signaling cascade which
promotes the phosphorylation and nuclear translocation of IRF3, and subsequent induction of type
I/III interferon-stimulated genes (ISG). This overall results in cytokine production which favors
the recruitment of TILs. Aberrantly expressed ERV may also contribute to the development of an
adaptive immune response through the production of tumor associated neoantigens (TAA). Upper
left part of the tumor cell: in RMC and within a context of MYC-induced replication stress, dsDNA is
released in the cytoplasm, which activates the cGAS/STING DNA-sensing pathway. The DNA sensor
cGAS binds to dsDNA, which triggers the formation of cyclic G-AMP (cGAMP), and subsequently
activates STING. cGAMP-bound STING recruits TBK1 and phosphotylates IRF3, which translocates
to the nucleus where it triggers the expression of ISG.

Biomedicines 2022,10, 650 6 of 17
Chun et al. further discovered that MRT and the ATRT-MYC subgroups presented a
immunologically active microenvironment, with increased cytotoxic T-cell infiltration [
72
].
Interestingly, they did not evidence ERV de-repression, but identified nine aberrantly
expressed tumor antigen genes, whose expression was specific to MRT and associated with
increased T-cell infiltration.
In RMC, Msaouel et al. identified that the dsDNA–sensing cGAS/STING pathway
was activated and associated with enhanced tumor immunogenicity (Figure 2) [
73
]. They
suggested that SMARCB1 loss resulted in enhanced replication stress following the activa-
tion of the c-MYC pathway, and subsequent increased cell-cycle checkpoint activation and
DNA damage. However, how exactly SMARCB1 loss leads to cGAS/STING activation,
and whether this is also the case in other SMARCB1-defective carcinomas or sarcomas,
remains to be elucidated.
3.2. SMARCB1-Deficient Tumors’ Immune Microenvironment
3.2.1. Tumor-Infiltrating Lymphocytes (TILs)
The tumor immune microenvironment of pediatric cancers has traditionally been
anticipated to be poorly infiltrated or immune-excluded [74], notably as a consequence of
their simple genome and low TMB. Surprisingly, despite sharing the same unique genomic
alteration, rhabdoid tumors are molecularly heterogeneous [
75
] at the epigenomic and
transcriptomic level.
By studying the DNA methylation, Chun and colleagues identified five MRT sub-
groups: (1) ATRT-MYC, (2) ATRT-TYR, (3) RTK, (4) ERRT, and (5) ATRT-SHH [
72
]. Groups
1, 3, and 4 presented a high expression of immune-related genes, notably those involved
in antigen processing, antigen presentation, T-cell activation and homing, and innate im-
mune response. This correlated with increased infiltration of CD8+ cytotoxic T-cells by
immunohistochemistry and enriched immune-related gene signatures in RNA-Seq, along-
side with increased infiltration of PD-L1+ CD68+ myeloid cells [
72
]. Similarly, Leruste
and colleagues found that the ECRT and ATRT-MYC subgroups were highly infiltrated by
CD8+ T-cells. Using single cell RNA and T-cell receptor sequencing, they identified clonally
expanded resident memory CD8+ T-cells, suggesting the presence of a local tumor-specific
anti-tumor immune response [
66
]. In mouse syngeneic models, MRT were highly infiltrated
in
PD-1+
TILs and were highly sensitive to anti-PD-1 therapy (75% of complete response
and survival benefit).
Despite its low TMB, RMC is also highly infiltrated by TILs, myeloid dendritic cells,
neutrophils, and B-cells [
73
]. This overall suggests that, contrary to what was initially
anticipated, certain subgroups of tumors with simple genomics are immunogenic and may
benefit from immune therapy-based therapeutic approaches.
3.2.2. Tertiary Lymphoid Structure (TLS)
TLS are heterogeneous aggregates of B- and T-cells, forming follicle-like structures
containing a network of CD21+ and/or CD23+ follicular dendritic cells [
76
]. In breast,
colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer (NSCLC), and GIST,
the presence of TLS was found to be associated with a favorable prognosis [
77
–
81
]. In
NSCLC and melanoma, TLS-rich tumors are more infiltrated by CD4+ and CD8+ cells,
supporting a role for TLS in modulating T-cells in the TME even outside TLS [
82
,
83
].
Interestingly, the presence and density of TLS correlate with therapeutic responses to ICI in
several tumor types including frequent soft-tissue sarcomas (NCT02301039) [
84
], making
it an additional important predictive biomarker [
76
]. The presence of TLS in SMARCB1-
defective sarcoma and its association with response to immunotherapy have not been
studied yet.
3.2.3. Myeloid Populations
Beyond T-cells, many other immune cells affect tumor response to immune therapies.
In particular, tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells

Biomedicines 2022,10, 650 7 of 17
(MDSCs) have been associated with poorer response to ICI [
85
,
86
]. Despite their potential
sensitivity to anti-PD-(L)1 therapy, MRT are also infiltrated by myeloid cells, including
monocytes, macrophages with pro-tumoral M2-like signatures [
66
,
72
]. Recent studies have
also reported a predominant infiltration of M2-like TAMs in ES and chordoma [87].
3.2.4. Immune Checkpoint Inhibitors
Anti-PD-(L)1 therapy is the most frequently used ICI. High PD-L1 expression, using
FDA-approved immunohistochemistry companion diagnostic tests (e.g., 22C3 pharmDx
on Dako Omnis, Agilent, for pembrolizumab, MSD; or SP263 on Ventana for atezolizumab,
Roche), is currently the most robust predictive biomarker of response to anti-PD-(L)1 agents,
and is routinely used in patients with advanced NSCLC, bladder cancer, and head and
neck squamous cell carcinoma [59,88–92].
MRT present a variable PD-L1 expression [
93
,
94
]. Abro and colleagues found that eight
(out of 16) cases of ATRT/MRT were PD-L1-positive (tumor proportion score: 10–70%), and
that nine cases displayed high PD-1 expression in TILs [
93
]. However, PD-1/-L1 expression
was not predictive of survival. More recently, Forrest and colleagues investigated PD-L1
expression in 30 SMARCB1-negative sarcomas, including ES and anaplastic chordoma [
94
].
They found that 47% of cases were PD-L1 positive (
≥
1% of positivity in tumor cells
or TILs), and that this was more frequent in extracranial sites, with 4/4, 4/9, 1/9, and
1/1 in ES, ERRT, ATRT, and anaplastic chordoma being PD-L1 positive. Using TCGA
RNA-sequencing for analysis of tumor-infiltrating immune cells, they observed an inverse
correlation between SMARCB1 mRNA levels and both CD8 and PD-L1 expression across
multiple cancer types [
94
]. PD-L1 expression has also been explored in larger cohort of
various sarcoma subtypes (including ES), with expression varying from 0 to 100% [
94
–
97
].
Interestingly, PD-1 seemed to be highly expressed when PD-L1 was absent [
97
]. In the study
from Kim et al., where 7/7 ES were PD-L1 high (using a threshold of >10% of tumor cells
for positivity), PD-L1 expression was significantly associated with shorter 5-year overall
survival and an independent negative prognostic factor, thereby supporting its role as a
potential therapeutic target. High PD-L1 levels have also been reported in chordoma, where
it correlated with the presence of TILs, metastatic status, and worse prognosis [98,99].
Beyond PD-1/-L1, Leruste et al. reported that other immune checkpoint receptors
(including HAVCR2/TIM-3 and LAG3) were expressed by CD8+ T-cells in extra-cranial RT
and MYC-AT/RT, suggesting that T-cell exhaustion may contribute to immune escape in
these diseases [66].
4. Clinical Efficacy of Immune Therapies in SMARCB1-Defective Tumors
Here, we will focus on the results of clinical trials evaluating the immune checkpoint
inhibitors targeting the PD-1/PD-L1 and CTLA-4/CD80/86 checkpoints, as these are the
only immune therapies evaluated in the clinic in these diseases so far.
4.1. Anti-PD(L)-1 Therapy as a Monotherapy
Clinical benefits of immune checkpoint inhibitor therapy in SMARCB1-negative sar-
coma have been reported in several clinical trials and individual case reports (Table 2).
Regarding clinical trials, five partial responses have been reported out of nine patients with
MRT or ES treated with anti-PD(L)1 [
100
–
103
]. Three responses were long-lasting, with
durations of 12, 13, and 18 months [
100
,
101
]. Forrest and colleagues further reported some
clinical benefit of anti-PD-(L)1 therapies in three pediatric patients with SMARCB1-negative
diseases [94]. The first patient, who was treated as first-line therapy for an ES (PD-L1+ on
40% of the tumor cells), presented a prolonged stable disease and stayed 12 months on
therapy; the second patient with PD-L1+ (5% of tumor cells) anaplastic chordoma presented
a shrinkage of some lesions and overall stable disease for 9 months on nivolumab; the last
patient received pembrolizumab as first-line treatment for a MRT and presented a stable
disease during 15 weeks. TLS were not evaluated in these cases.

Biomedicines 2022,10, 650 8 of 17
Table 2. Results of clinical studies evaluating ICI in monotherapy in SMARCB1-deficient sarcoma.
Reference
NCT
Identifier/
Trial Name
Study
Design Study Description Number
of Patients
Specific
Histotype
Best
Response
Duration
of Best
Response
Paoluzzi,
2016
Retrospective
series
Nivolumab in relapsed
metastatic/unresectable sarcomas 2ES 1 PR 3.8 mth
1 PD
Blay, 2019
NCT03012620/
AcSéPhase II Pembrolizumab for patients with selected
rare cancer types 1 MRT PR NA
Georger,
2020
NCT02541604/
iMATRIX Phase I/II
Atezolizumab in children and young
adults with refractory or relapsed solid
tumors, with known or expected
PD-L1 expression
3 MRT PR NA
Georger,
2020
NCT02332668
Phase I/II
Pembrolizumab in pediatric patients with
PD-L1-positive, advanced, relapsed, or
refractory solid tumor
2 MRT 1 PR 17.8 mth
1 PD
1 ES PR 11.8 mth
Forrest,
2020 Case
report Pembrolizumab 1 ES SD 12 mth
Nivolumab
Pembrolizumab
1 PDC PR 9 mth
1 MRT SD 15 wk
Abbreviations: ES: epithelioid sarcoma; MRT: malignant rhabdoid tumor; PDC: poorly differentiated chordoma;
PR: partial response; PD: progressive disease; SD: stable disease; NA: not available.
4.2. Anti-PD-(L)1 Therapy in Combination
Several combinatorial strategies have been proposed in order to improve the efficacy
of ICI and fully unleash their antitumor potential (Table 3). These include combinations
with other ICI, targeted therapies, anti-angiogenic agents or epigenetic drugs which all aim
at synergistically boosting the anti-tumor immune response [6,104].
Table 3.
Results of clinical studies evaluating ICI therapy in combination in SMARCB1-deficient sarcoma.
Reference
NCT
Identifier/Trial
Name
Study
Design Study Description Number
of Patients
Specific
Histotype
Best
Response
Duration
of Best
Response
D’Angelo, 2018
NCT02500797/
Alliance
A091401 Phase II Nivolumab with or without
ipilimumab treatment for
metastatic sarcoma
1 ES 0
Wilky, 2019 NCT02636725 Phase II Axitinib + pembrolizumab in
advanced sarcoma 1 ES PR 24 wk
Martin-Broto,
2020 NCT03277924
Phase Ib/II
Nivolumab and sunitinib
combination in advanced soft
tissue sarcomas
7 ES SD 17 mth
D’Angelo, 2017
NCT01643278 Phase Ib
Combined KIT and CTLA-4
Blockade in patients with Refractory
GIST and other advanced Sarcomas:
Ipilimumab + dasatinib
1 ES SD 16 wk
Pecora, 2020 Case
report Nivolumab + ipilumab 1 ES Complete
response NA
Abbreviations: ES: epithelioid sarcoma; PR: partial response; SD: stable disease; NA: not available.
4.2.1. Dual ICI Combination
Combined targeting of the anti-PD-1 nivolumab with or without the anti-CTLA4
ipilimumab in metastatic sarcoma has been first evaluated in the randomized clinical
trial Alliance A091401 (NCT02500797) [
105
]. Across all sarcoma subtypes, the combined
administration resulted in an objective response rate of 16% (6/38 patients) and median
progression-free survival of 4.1 months, versus 5% (2/38 patients), and 1.7 months with
nivolumab monotherapy; the only patient with ES enrolled in this trial received nivolumab
monotherapy and did not respond to treatment. By contrast, Pecora and colleagues re-
ported a rapid and complete response to nivolumab and ipilimumab in a chemotherapy-
and tazemetostat-pretreated patient with advanced SMARCB1-negative ES [
106
], calling for
further molecular investigation of the determinants of response to ICI in this context. The
nivolumab plus ipilimumab combination is currently being further evaluated in metastatic
sarcoma of rare subtype including ES and chordoma (NCT04741438) (Table 4). Additionally,

Biomedicines 2022,10, 650 9 of 17
based on the preclinical evidence that supports the presence of an immunogenic microenvi-
ronment in SMARCB1-defective tumors [
94
], a dedicated phase II clinical trial evaluating
the efficacy of nivolumab and ipilimumab SMARCB1-negative pediatric cancers has been
launched (NCT04416568). Similarly, a phase II trial evaluating nivolumab plus ipilimumab
in patients with SMARCB1-deficient renal malignancies is ongoing (NCT03274258). We
can hope that these trials will bring interesting and more homogeneous results, to better
understand the role of SMARCB1 in tumor immunogenicity and optimize the use of ICI in
patients with SMARCB1-negative diseases.
Table 4. Selected ongoing immunotherapy-based clinical trials for SMARCB1-deficient sarcoma.
NCT
Identifier/
Study Name
Drugs
Clinical
Trial
Phase
Population Estimated
Enrollment
Primary
Completion Date Location
NCT04741438/
RAR-Immune Nivolumab +
Ipilimumab III
Metastatic or unresectable
advanced sarcoma of rare subtype
including ES and chordoma
96 February 2025
Centre Léon
Bérard, Lyon,
France
NCT04416568 Nivolumab +
Ipilimumab II
Relapsed or refractory
INI1-negative cancers in children
and young adults from 6 months
to 30 years
45 October 2023
Dana-Farber
Cancer Institute,
United States,
Massachusetts
NCT04705818/
CAIRE Durvalumab +
Tazemetostat II
Distinct cohorts of solid tumors
including soft-tissue sarcoma and
metastatic solid tumor with
positive interferon gamma
signature and/or presence of TLS
173 October 2022 Multiple French
Cancer Institute
NCT04095208/
CONGRATS Nivolumab +
Relatlimab II
Advanced
non-resectable/metastatic soft
tissue sarcoma with high-level of
tertiary lymphoid structures
67 March 2022 Multiple French
Cancer Institute
Abbreviations: ES: epithelioid sarcoma; TLS: tertiary lymphoid structure.
4.2.2. Anti-Angiogenic Agents and ICI
Combination with antiangiogenic therapeutics targeting VEGF or VEGF receptor
(VEGFR) has been supported by pre-clinical evidence that VEGF inhibits T-cell development
and may contribute to tumor-induced immune suppression [
107
]. Interestingly, a prolonged
(24 weeks) objective response has been reported in the only patient with ES enrolled in the
clinical trial evaluating the axitinib and pembrolizumab combination [
108
]. Intriguingly,
and by contrast, no objective response was observed among the seven patients enrolled
in the phase Ib/II trial evaluating combination of nivolumab with sunitinib [
108
,
109
].
Similarly, the combination of dasatinib (a multikinase inhibitor with an primarily anti-src
family kinase activity) and ipilimumab did not show any efficacy in a patient with ES [
110
].
4.2.3. Epigenetic Modulators and ICI Combinations
Multiple preclinical studies support that epigenetic modulators—and, notably, EZH2
inhibitors—have the potential to modulate tumor’s immunogenicity and anti-tumor im-
mune response [111].
EZH2, a histone methyltransferase, is the core enzymatic subunit of the PRC2 complex.
EZH2 catalyzes the methylation of lysine 27 on histone H3 (H3K27me3) at promoters and
enhancers, leading to the epigenetic silencing of genes notably involved in cell fate and
proliferation [
112
]. In normal cells, mSWI/SNF complexes oppose to PRC2 transcriptional
repression. This epigenetic antagonism has been best characterized in SMARCB1-deficient
MRT models where loss of SMARCB1 is associated with constitutive EZH2 activation
and downstream oncogenic activity; in MRT preclinical models, EZH2 inhibition induced
durable tumor regression and apoptosis [
113
–
115
]. These preclinical results have supported
the development of Phase II basket multicenter clinical trial evaluating the EZH2 inhibitor
tazemetostat in patients with SMARCB1-negative solid tumors (NCT02601950). On the
basis of the results of ES cohort [
43
] which showed a response rate of 15% and some
degree of tumor shrinkage or prolonged stabilization in more than half of the patients, the

Biomedicines 2022,10, 650 10 of 17
Food and Drug Administration (FDA) recently approved tazemetostat for the treatment
of metastatic or locally advanced ES [
116
]. Beyond ES, EZH2 inhibitors have also shown
efficacy in other SMARCB1-defective diseases in children, such as chordoma or ATRT, with
50% and 19% response rates, respectively [117].
Beyond their direct action through epigenetic antagonism, EZH2 inhibitors could
also be used as immunomodulators, and the role of EZH2 in the modulation of the tumor
immunogenicity has been extensively reviewed elsewhere [
111
,
118
]. In a nutshell, EZH2
inhibition can directly influence immunogenicity of cancer cells by (i): acting on the antigen
presentation process: EZH2 expression contributes to the downregulation of Major Histo-
compatibility Complex (MHC)-I and MHC-II, which subsequently dampens the anti-tumor
immune response [
119
,
120
]; (ii) activating the cell-autonomous innate immune signaling
pathways: in chemo-resistant small cell lung cancer cells, inhibition of EZH2 favored the
de-repression of the SPARCs, a subclass of ERVs, which triggered the activation of the type
I IFN pathway [
121
]; (iii) rewiring the tumor immune microenvironment: EZH2 inhibition
has been reported to result in the reactivation of TH1 cell-type cytokine expression, CXCL9
and CXCL10 and to increase infiltration of CD8+ T cells in human ovarian cancer mod-
els [
122
]. Through these various actions, EZH2 is both involved in the primary sensitivity
to ICB, and in adaptive resistance mechanisms [
120
]. Multiple studies have reported that
pharmacological inhibition of EZH2 is able to circumvent primary or acquired resistance to
anti-PD-(L)1 resistance in several cancer
types [120,123–125]
. In line with these preclinical
results, Gounder et al. reported a prolonged (>2 years) and exceptional abscopal response
to radiotherapy in a patient with a SMARCB1-negative poorly differentiated chordoma
who previously progressed on the EZH2 inhibitor tazemetostat [
43
]. Comparisons of tumor
biopsy samples obtained prior to and during tazemetostat revealed a substantial increase
in intra-tumoral and stromal infiltrates of CD8+ cytotoxic and FOXP3+ regulatory T cells,
together with an enhanced expression of the PD-1 and LAG3 immune-checkpoint proteins
on T cells. The ongoing multicenter phase II CAIRE clinical trial (NCT04705818, Table 4),
which evaluates the association of anti-PD-L1, durvalumab and the EZH2 inhibitor tazeme-
tostat in solid tumors including soft-tissue sarcoma, will shed light on the potential of this
combination and results are eagerly awaited.
5. Perspectives and Future Directions
5.1. SMARCB1: A Role Which Is still Unclear in Modulating Tumor Immunogenicity
As described above, multiple converging preclinical data support that loss of SMARCB1
leads to a more immunogenic tumor microenvironment, at least in some tumor types.
Though, several aspects are still unclear and deserve further investigation.
First, SMARCB1-deficient tumors encompass multiple various neoplasms in different
anatomic sites. In some of these, such as MRT, SMARCB1 is virtually the only genetic
alteration, but in others, such as ES, the tumor genome is much more complex. Additionally,
the mechanisms by which SMARCB1 loss may favor tumor immunogenicity can differ,
as described above in MRT and RMC, suggesting some variability according to the cell
of origin [
66
,
73
]. How these differences influence tumor immunogenicity is still unclear.
Collaborative efforts, notably aiming at comparing the immune profiles of various diseases
belonging to the “SMARCB1-deficient spectrum” of malignancies, would bring very useful
insight on this question.
Similarly, even within a given SMARCB1-defective histological type, heterogeneity of
immune infiltration has been reported, as illustrated by the various AT/RT methylation
subgroups [
72
]. Deeper molecular characterization of larger series (including not only
RNA sequencing but also methylome analysis, multiplex immunohistochemistry to define
immune populations, and TLS assessment), should further shed light on this.
Finally, clinical results of ICI therapy efficacy in SMARCB1-defective sarcoma are still
scarce due to the rarity of these diseases, and the heterogeneity of the available results
in terms of patients’ characteristics (various histologic subtypes, treatment lines, etc.).
Ongoing clinical trials will hopefully bring more homogeneous results and be associated

Biomedicines 2022,10, 650 11 of 17
with insightful translational studies which will eventually help in understanding the role
of SMARCB1 defect in tumor immunogenicity.
5.2. Improving Patient Selection for Immune Checkpoint Inhibitors
Beyond SMARCB1, several important biomarkers should also be taken into account
when selecting patients for immune checkpoint blockers targeting the PD-1/anti-PD-L1.
For example, TLS were not assessed in studies that evaluated ICI in SMARCB1-deficient
sarcoma so far. Additionally, transcriptional signatures which predict response to anti-PD-
(L)1 therapies, such as an active IFN-
γ
signature, may further help identifying patients
who are most likely to benefit from ICI therapy [
126
,
127
]. As RNA sequencing is becoming
more and more widely used in sarcoma diagnostic and more generally in routine tumor
profiling, this information should be exploited as well.
5.3. Other Immunotherapeutic Approaches
Other immune checkpoint inhibitors are currently being evaluated, such as relatlimab.
This monoclonal antibody targeting lymphocyte activation gene-3 (LAG-3) is evaluated
in combination with nivolumab in a phase II clinical trial enrolling patients with selected
TLS-positive sarcoma (NCT04095208) (Table 4). Further, T-cell-based approaches (including
CAR-T cells and genetically engineered TCRs) may represent a promising approach to
target antigens highly or specifically expressed in certain types of SMARCB1-defective
diseases. For example, preclinical
in vivo
studies in mice show that CAR-T cells directed
against the B7/H3 checkpoint trigger a potent antitumor immune response with complete
clearance of cerebral ATRT xenografts, when administered intracerebroventricularly or
intratumorally [128].
6. Conclusions
Converging recent data support that SMARCB1 loss favors anti-tumor immunogenic-
ity, at least in certain subtypes of SMARCB1-deficient sarcomas and carcinomas. Whether
this can be generalized SMARCB1-deficient diseases remains an important and open ques-
tion. Mechanisms by which SMARCB1 modulates tumor immunogenicity are diverse and
potentially disease-specific, which suggests that clinical therapeutic approaches should
be customized accordingly. Because SMARCB1-defective tumors are rare, clinical cases of
response to immune checkpoint inhibitors are still anecdotal, but clinical trials are ongoing
and series expanding rapidly. We can hope that the ongoing collaborative efforts on pre-
clinical studies in SMARCB1-defective models and on the molecular characterization of
SMARCB1-defective tumors, together with the growing results of several ongoing clinical
studies, will allow to unravel the molecular determinants of response to immunotherapy
in SMARCB1-deficient tumors. At a time where multiple forms of immune therapies are
being development at an unprecedented speed, either as a monotherapy or in combination
with other anti-tumor agents, this brings an unprecedented hope for the treatment of these
deadly diseases and opens novel therapeutic avenues for patient’s benefit.
Author Contributions:
Writing—original draft preparation: C.N. and S.P.-V.; writing—review and
editing, C.N. and S.P.-V. All authors have read and agreed to the published version of the manuscript.
Funding:
No funding has been received directly linked to this manuscript. As part of independent
research on epithelioid sarcoma, Sophie Postel-Vinay has received funding from the Cancéropôle
Ile-de-France and La Ligue Contre le Cancer.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

Biomedicines 2022,10, 650 12 of 17
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