Facts and Hopes in Immunotherapy of Soft Tissue Sarcomas

Abstract

Sarcomas are mesenchymal tumors, encompassing more than 175 subtypes, each one with their own genetic complexities. As a result, immunotherapy approaches have not been universally successful across the wide rage of diverse subtypes. The actual state of science and the current clinical data utilizing immunotherapy within the soft tissue sarcomas (STS) will be detailed in this review. More precisely, the review will focus on: a) the role of the immune microenvironment in the development and activity of new therapeutic approaches; b) the recent identification of the sarcoma immune class (SIC) groups, especially group SIC E with its B cell signature that predicts immunotherapy response; c) the clinical trials using PD-1 and/or CTLA-4 inhibitors, which serves as reference for response data, d) the promising clinical activity from the combination of anti-angiogenics agents with PD-1 inhibitors E) the adapted T-cell therapies for synovial sarcoma that target either NY-ESO or MAGEA4; and F) the role for localized therapy using the virotherapy T-VEC with PD-1 inhibitors. Herein we present the facts and the hopes for the sarcoma patients, as the field is rapidly advancing its understanding of what and where to use the various types of immunotherapies.

Introduction

Immunotherapy for sarcoma has its origins in 1891 with William B Coley injecting mixed toxins of the Streptococcus erysipelas and the Bacillus prodigiosus into sarcoma patients(1). These early experiments demonstrated that immunotherapy was a possibility for the treatment of sarcomas. The early promise of immunotherapy was then placed on hold for over a century (2). In the interim, a revolution in our understanding of the genetics of STS occurred that demonstrated the diversity and complexity of different types of STS (3,4). It is the same genetic complexity that has complicated a one size fits all immunotherapy approach for STS. In this review, we will discuss the current advances in our understanding of sarcoma immunobiology and the effects that immunotherapy has had in clinical trials. From the facts of where we currently stand, to the hope that exists for immunotherapy in STS, we are still very much at the beginning of the beginning.

The Microenvironment of STS

The immune microenvironment in STS substantially differs from other tumors where immunomodulation effectively functions, such as in the case of melanoma. When compared to immune responsive tumors, STS demonstrated a median tumor mutational burden (TMB) of 2 mutations per DNA megabase (Mb)(5), CD8⁺ expression was 23±13 %(6) and the PD-L1 expression was 6.6% in the largest series (7). In contrast, the expression in melanoma, a paradigmatic immune sensitive tumor, was 14 mutations per DNA Mb in TMB, 42 ± 23 % for CD8⁺ lymphocytes(8) and 35% for PD-L1 (9).

Sarcoma genomic heterogeneity complicates these statistics. The highest range of TMB expression is found in cutaneous angiosarcoma (10), undifferentiated pleomorphic sarcoma (UPS), leiomyosarcoma, sarcoma not otherwise specified (NOS) and myxofibrosarcoma with median TMB ranging from 2.2 to 401.4 mutations per DNA Mb. On the opposite end of the spectrum, synovial sarcoma, myxoid liposarcoma, solitary fibrous tumor or alveolar rhabdomyosarcoma exhibit lower TMB, with a median of 1.7 and maximum TMB ranging from 7.5 to 28.4 mutations per DNA Mb.(5)

Mismatch repair (MMR) deficiency occurs by hypermutation of MMR genes, germline MMR pathway mutation or double somatic mutation in MMR genes and this is termed microsatellite instability (MSI). MSI-high phenotypes have the highest response probability to PD-1 inhibitors.(11) Sarcomas are rarely MSI-high (0.78%)(12), though patients should still be tested for this status.

Immune Infiltrates in Sarcoma Microenvironment

Studies characterizing infiltrates of immune cells in sarcoma show a low number of tumor infiltrating lymphocytes (TILs) compared to melanoma. The mean and standard deviation of number of cells per gram of weight were as follows: 72±15 for CD3⁺ and 42±23 for CD8⁺ in melanoma while there were 35±9 for CD3⁺ and 23±13 for CD8⁺ in sarcoma, respectively.(6) In a meta-analysis carried out to examine the prognostic role of high TIL population in different cancers, including 52 studies and 12447 patients, the authors reported a positive effect on prognosis for high CD3⁺ and CD8⁺ TILs. The CD8/Foxp3 ratio was the best prognostic indicator of risk of death with HR of 0.48 (95% CI 0.34–0.68).(13) In contrast, in series including several STS subtypes, the prognostic role of CD3⁺ or CD8⁺ seems less prominent. In a series with 249 patients with STS, 83% of them localized, high number of CD20⁺ lymphocytes in tumor significantly correlated with a longer disease specific survival. In multivariate analyses, high number of CD20⁺ lymphocytes was the only independent prognostic factor among TIL for disease free survival.(14)

The prognostic role of CD8⁺ is unclear, as in a series of 163 STS (81% localized at diagnosis) where high CD8⁺ lymphocytes (cut-off 137 cell/mm²) significantly correlated with poor disease free survival (p=0.031) and overall survival (p<0.001) in the univariate analysis.(15) This inconsistent outcome could be related to the high heterogeneity inherent to the inclusion of several STS subtypes in this study. Nevertheless, a few studies have focused on the prognostic impact of TIL in specific histotypes; however, with inconsistent results. Contradictory findings were seen for example in synovial sarcoma: while in one study with 36 synovial sarcoma patients high CD8⁺ or Foxp3 lymphocytes correlated with better prognosis,(16) another study including 22 synovial sarcoma patients showed worse prognosis for patients with high CD8⁺ lymphocytes.(17) Additional series exploring the prognostic role of TIL in malignant peripheral nerve sheath tumors (18) and cutaneous angiosarcoma(19) also showed divergent prognostic outcome. Besides, the largest series exploring the prognostic impact of TIL in sarcoma, included 809 samples of STS and GIST, did not find any prognostic correlation among the translocation-associated sarcomas. By contrast the authors reported a significant better prognostic for OS (p=0.02) and PFS (p=0.01), with increasing lymphocyte infiltration among the non-translocation-associated sarcomas. Additional, the OS was significantly worse with increasing CD56⁺ (p=0.03) or PD-1⁺ (p=0.05) TIL.(20) Of note, patients with positive immunostaining for both CD8 and FOXP3 had better OS compared with those negative for FOXP3 in dedifferentiated liposarcoma (p=0.002) or MPNST (p=0.002). In myxoid liposarcoma this correlation was inverse (p<0.001).(20)

Lymphocytic infiltrates has been explored also by gene expression. Sarcomas with a complex genome expressed high levels of genes related to T-cell infiltration and antigen presentation. For instance, CD3 and IL-7 receptor (CD127) expression was significantly higher in leiomyosarcoma and UPS than in translocation-related sarcomas. A trend toward higher expression in non-translocation related sarcoma was also noted for IDO, CD4, CD27 and CCR5.(21) In addition, TCR clonality has been found to be correlated to PD-1 (p=0.007) and PD-L1 (p=0.003) expression (21). Immune signatures characterizing the type of immune infiltration in different sarcomas were analyzed based on TCGA mRNA data using 203 genes involved in immune response. Thus, UPS and myxofibrosarcoma had the highest median macrophages scores, dedifferentiated liposarcoma had highest CD8⁺ scores and somatic leiomyosarcoma exhibited the highest PD-L1 score.(4) Of note, macrophages, monocyte-derived phagocytic cells, play a crucial role in tumor immunomodulation. Tumor-associated macrophages (TAMs) can mediate for anticancer effects or for tumor progression depending on their polarization. In general, M1-polarized macrophages mediates anticancer effects through adaptive immunity mechanisms and M2-polarized macrophages suppress adaptive immunity, favoring tumor progression, tumor angiogenesis, increase extracellular matrix breakdown and tumor invasion.(22) TAMs have been associated with poor survival in myxoid liposarcoma,(23) gynecologic(24) and non-gynecologic leiomyosarcomas,(24,25), solitary fibrous tumor(26) and UPS.(27) Targeting the colony-stimulating factor 1 receptor (CSF1R), a protein that facilitates the differentiation of monocytes into TAMs and promotes their survival within the tumor, with CSF1R inhibitors(28,29) could be a therapeutic strategy to be considered for these histologic subtypes in future trials. Natural killer (NK) cells were the only cell type to correlate significantly with disease specific survival. Table 1 lists studies examples of studies that analyzed prognostic impact of immune infiltrates in STS.

Table 1.

Examples of studies analyzing the expression of immune cells in the sarcoma microenvironment by immunohistochemistry and RNA signatures.

Study	N	Main subtypes	Stage	IHC	Clinical Endpoint	Independent Prognostic role
PROTEIN EXPRESSION
Sorbye(14)	249	STS	Localized 83%	CD3, CD4, CD8, CD20, CD45	DSS (better prog)	High CD20+
Que(15)	163	STS	Localized 81%	CD3, CD4. CD8, LAG3	DFS, OS (worse prog)	High CD8+ and LAG3 (univariate)
Oike(16)	36	Synovial sarcoma	Localized 92%	CD4, CD8, Foxp3, CD163	OS (better prog) PFS/OS (worse prog)	High CD8 or Foxp3 (Univariate) High CD163 (univariate)
Van Erp(17)	22	Synovial sarcoma	Localized 50%	CD8	MFS (worse prog)	High CD8 (univariate)
Fujii(19)	40	Cutaneous angiosarcoma	Localized	CD4, CD8, Foxp3, MHC-I,	OS (better prog)	High CD8+ (univariate)
Shurell(18)	38	MPNST	Localized 92%	CD8	DSS/DFS	No correlation
Dancsok(20)	809*	STS and GIST	UNK	CD4, CD8, CD56, FOXP3, PD-1, PD-L1, TIM-3, Lag3	OS/PFS** (better prog) OS** (worse prog)	High TIL (multivariate) CD56+ or PD-1+ TIL (multivariate)
Rusakiewicz(63)	57	GIST	Localized	CD3, Foxp3, NKp46	PFS (better prog)	NKp46, CD3 (multivariate)
Zheng (64)	72	STS	Localized and Recurrent	CD8, PD-L1, CD20, FOXP3	OS	High CD8 (univariate)
Petitprez (55)	589	STS	Localized	CD3,CD20,PD1 CD21,CD23, CXCR5, CD21, CD4,	OS (Signature of bettter and worse prog)	CD20 (multivariate) High TIL
mRNA EXPRESSION
Study	N	Main subtypes	Stage	Genes	Clinical Endpoint	Independent Prognostic role
Abeshouse(4)	206	STS	UNK	203 genes	DSS	NK (univariate)
Neo (65)	259	STS	Localized	CD73 and NK cell signature	OS	No correlation

DFS (disease free survival); OS (overall survival); DSS (disease-specific survival); MPNST (malignant peripheral nerve sheath tumors); MFS: metastases-free survival.

^*The study also included 263 bone sarcomas;

^**in non-translocation-associated sarcomas.

Sarcoma Immune Classes

Using a transcriptomic analysis of the microenvironment cell population, which measure the expression of 8 immune and 2 stromal cell populations,(30) STS can be classified into five different sarcoma immune classes (SIC). Each SIC exhibited a different profile, from A (immune desert) which showed the lowest expression of gene signatures of immune cells and vasculature expression, to E (immune and tertiary lymphoid structures) characterized by the highest expression of genes related to immune cells. In the middle, C (vascularized) was characterized by a high expression of endothelial related genes. SIC B and D have expressed mixed profiles between A and C or C and E. Of note, grouping sarcomas into these five classes based on different profile expression of tumor microenvironment resulted in prognostic impact. Thus, patients with SIC A showed worse overall survival than SIC D (p=0.048) or SIC E (p=0.025). Furthermore, this genomic immune signature had predictive role in a prospective series treated with pembrolizumab. The overall response rate (ORR) was 50%, 25%, 22%, 0% and 0% for SIC E, D, C, B and A respectively. Patients harboring SIC E had significantly higher ORR with pembrolizumab (p=0.026). A more detailed analysis revealed a significant correlation of survival with B-cell lineage signature, whereas CD8⁺ signature did not significantly correlate with survival.

Immune Checkpoints in Sarcoma Microenvironment

Specific immune checkpoint expression as PD-1/PD-L1 axis, have not demonstrated convincing prognostic or predictive value in sarcoma. In one study with 105 (74% localized) STS patients expressing intratumor PD-L1 in 65% of cases and a worse prognosis was observed. The expression of tumoral PD-L1 predicted for shorter overall survival (HR 5.69, 95% CI 2.558–12.700; p< 0.001) and event free survival (HR 3.27, 95% CI 1.776–6.036; p<0.001).(31) However, in other studies including different sarcoma subtypes, no prognostic correlation could be established.(21,32,33) Moreover, there were substantial differences for the same study among cases studied by tissue micro-array or by whole sections from the block.(34) Thus, the predictive value of PD-L1 expression remains uncertain, and caution should be used in clinical practice when utilizing expression to make theraputic off label recommendations.

A genomic approach, addressing the gene expression of PD-L1, could circumvent the constraints of PD-L1 immunohistochemistry. In one genomic array performed on 758 previously untreated sarcoma samples, 470 of them considered in the prognostic analysis, authors reported a significant correlation with metastasis free interval (MFS). PD-L1 high expression had 5-year MFS of 61% (95% CI 50–73) while PD-L1 low expression had 5-year MFS of 72% (95% CI 63–83), p=0.0037. This prognostic value has been confirmed in a validation set and PD-L1 expression had an independent prognostic value in the multivariate analysis, HR 1.51 (95% CI 1.06–2.16), p=0.024.(35) In TCGA analysis the highest PD-L1 expression was observed in leiomyosarcoma (4), while in another study, the highest expression was in UPS.(21) Additionally, a second study found significantly higher mRNA expression of PD-L1 in UPS.(36) Table 2 lists examples of studies analyzing prognostic impact of PD-L1 expression in tumor cell in the sarcoma context.

Table 2.

Examples of studies analyzing PD-L1 expression in tumor cells, by protein and RNA expression, in sarcoma and their prognostic role.

Study	N	Main subgroups	Stage	% Tumor Cells PD-L1	Prognostic correlation	Antibody
Protein expression (IHC)
Boxberg (66)	128	STS	Localized	28.1%	DFS and OS	Ventana
Botti (67)	24	angiosarcoma	Localized	66%	No	Ventana
Kim(31)	105	STS	Localized 74%	65%	EFS/OS Multivariate	Santa Cruz
Pollack(21)	81	STS	Localized 78%	59%	No	Merck Research
Dancsok(20)	809*	STS and GIST	UNK	22%	No	Ventana
D’Angelo(32)	50	STS & GIST	Localized 92%	12%	No	Dako
Oike(16)	39	Synovial Sarcoma	Localized 92%	0%	NA	Abcam
Park(34)	120	UPS/DDLPS	UNK	22% DDLPS 20% UPS	RFS/OS	Dako
Kösemehmetoğlu(68)	222	STS	UNK	15%	With high grade	Cell Signaling
Torabi(69)	160	LPS/Rhabdo	UNK	1.5% LPS 3% Rhabdo	UNK	Abcam
Toulmonde(33)	371	STS	Localized	19%	No	UNK
He (70)	21	Synovial Sarcoma	Localized and Metastatic	14.3	No	Ventana
Lee (71)	83	UPS	Localized	72.8	No	Ventana
Dancsok(20)	809*	STS and GIST	UNK	3% of translocation assocaited and 12% otherwise	No	Ventana
Vargas (72)	522	STS	Localized and Recurrent	13%	N.D.	Ventana
Que (73)	163	STS	Localized	11.7	DFS and OS	Cell Signaling
Orth (74)	225	STS	Localized	15.6	OS	Ventana
mRNA expression
Bertucci(35)	470	STS	Localized	PD-L1 high 41%	MFS Multivariate	NA

LPS (liposarcoma); UPS (undifferentiated pleomorphic sarcoma); Rhabo (rhabdomyosarcoma); RFS (relapse free survival); MFS (metastasis free survival); UNK (unknown).

Monotherapy with PD1 Inhibitors

The pioneer study was SARC028 phase II trial with pembrolizumab flat dose at 200 mg every 3 weeks, which was conducted in 12 academic centers in USA, and enrolled patients in two different cohorts, soft tissue and bone tumors. Among STS, the selected histologies chosen based on prevalence, were leiomyosarcoma, UPS, synovial sarcoma, and dedifferentiated/well differentiated liposarcoma. The main endpoint was investigator-assessed objective response by RECIST 1.1, considering 25% of overall response rate as clinically meaningful and if less than 10% as ineffective. The authors reported objective response in 7 of 40 (18%, 95% CI 7–33) patients accrued in STS cohort, with a median duration of response of 33 weeks. Responses were seen in 4 UPS, 2 dedifferentiated liposarcomas and 1 synovial sarcoma. The median of progression free survival (mPFS) and overall survival (mOS) for patients with STS were 18 weeks (95% CI 8–21) and 49 weeks (95% CI 34,73) respectively. The authors concluded that pembrolizumab was clinically active in patients with UPS and dedifferentiated liposarcoma.(37)

Tumor biopsies were required at baseline and after 8 weeks of treatment, which were crucial for gaining insight into the response to PD-1 blockade. Using a multiplex immunofluorescence including the following antibodies: PD-L1, CD3, CD8, PD-1, CD68, granzyme B, Foxp3, CD45RO, a correlation between response and expression of different immune cells or receptors was analyzed. Higher density of immune cells significantly correlated with response. PD-L1 expression in tumor cells were detected in only 2 of 40 analyzed cases (5%), and these were the two responding patients diagnosed with UPS. Given the lack of power within this study, only the only major conculsion was that more studies were warranted.

In the pharmacodynamics analysis comparing immune infiltrates from baseline and at week 8, the only remarkable changes were detected for two immune cell phenotypes: effector memory cytotoxic T-cells (CD3⁺ CD8⁺ CD45RO⁺) that increased from 7.9% to 21.5% and regulatory T-cells (CD3⁺ Foxp3⁺) or (CD3⁺ CD8⁺ Foxp3⁺), that increased from 3.7% to 8.3%. More interestingly, higher percentage of regulatory T-cells at baseline was correlated with a significant longer median of PFS: (40 vs 8 weeks, p=0.044), and similarly, higher percentage of cytotoxic T-cell infiltrates at baseline was correlated with a significant longer median PFS (40 vs 8 weeks, 0.016).

The study Alliance A091401, a non-comparative randomized phase II trial, randomized 85 patients to received nivolumab vs nivolumab plus ipilimumab in progressing STS patients after at least one previous systemic line. The main endpoint was investigator-assessed confirmed objective response by RECIST 1.1. Confirmed partial responses were observed in 2 of 38 evaluable patients (5%). Median PFS was 1.7 months (95% CI 1.4–4.3) and the estimated 6-month PFS rate is 15%. (38) Of note, the two arms of the trial were non-comparator by design, so direct comparison of both arms is not possible.

Combination therapies with immune checkpoint inhibitors

Because of poor results observed with monotherapy, investigators have explored combinations for a more efficient immunomodulation, trying to convert the sarcoma microenvironment into T-cell-inflamed tumor. Alliance A091401 is the only reported randomized trial testing nivolumab alone against ipilimumab and nivolumab in STS. There were 6 objective responses of 38 evaluable patients (16%; 92% CI 7–30) in the combination arm. Responses were seen in UPS (2), leiomyosarcoma (2), angiosarcoma (1) and myxofibrosarcoma (1). The mPFS was 4.1 months (95%CI 2.6–4.7) and the mOS was 14.3 months (95% CI 9–6-not reached). (38)

Angiogenesis mediators as VEGFA have a well-known function promoting neoangiogenesis while preventing immune response.(39) A phase 2 clinical trial examining Axitinib 5 mg twice daily with pembrolizumab 200 mg starting on day 8 and then every 3 weeks was investigated in 33 STS patients. There were 69% of patients with at least 2 previous lines, 51% that had received previous tyrosine kinase inhibitors and 36% diagnosed with alveolar soft part sarcomas (ASPS). The endpoint was 3-month PFS rate of 40%. The 3-month and 6-month PFS rate were 65.6% (95% CI 46.6–79.3) and 46.9 % (95%CI 29.2–62.8) respectively. Of 32 evaluable patients, 8 (25%) had partial response, and 9 (28%) had stable disease. Six responders were ASPS, one was epithelioid sarcoma and another was leiomyosarcoma. The median duration of response was 29 weeks while the mOS was 18.7 months. Neither PD-L1 positivity nor high TIL showed statistical correlation with PFS or partial response. Interestingly, angiogenic plasmatic activity at baseline was more likely to respond to this regimen.(40) Intriguingly, ASPS seems to be sensitive to immunotherapy-based regimens, even if this histologic subtype does not exhibit an immune sensitive microenvironment. In fact, it has been reported that ASPS has a TILs ratio lower than in non-translocation related sarcomas(20) and the TMB is also lower compared to other histologic subtypes, such as synovial sarcoma or ewing sarcoma(41). In this latter study, a mismatch-repair deficiency signature was associated with the activity of anti-PD-L1 in ASPS, however, further studied in larger series of cases are required to validate these observations. Besides, it is important to mention that some genes normally expressed in the context of ASPSCR1-TFE3 seemed to be involved in pathways of immune surveillance, immune-regulation by chemokines and focal adhesion. An example is CCL4, a gene positioned in the TFE3 neighboring cytogenetic band chr17q21 that signals through the receptor CCR5 and regulates, among other functions, macrophage migration.(42) This aspect could help understand in part the sensitivity of ASPS to anti-PD-1/PD-L1 inhibitors.

A similar approach used in the IMMUNOSARC trial, a phase I/II trial exploring the combination of sunitinib plus nivolumab in some STS and bone sarcoma patients. Data from phase II part of STS cohort was recently presented with 50 patients accrued. The recommended scheme derived after phase I part, was sunitinib 37.5 mg on a daily basis for the first 15 days, from then on nivolumab was administered at 3 mg/kg every 2 weeks and sunitinib dose was given at 25 mg per day. The main endpoint was 6-month PFS rate and the accrual was limited to UPS, synovial sarcoma, epithelioid sarcoma, angiosarcoma, clear cell sarcoma, extraskeletal myxoid chondrosarcoma, solitary fibrous tumors and ASPS. The reported 6-month PFS rate was 50% while the 6-month overall survival was 77% (median not reached). The response rate following central assessment was 11%, stable disease 61% and progressive disease 28%. ASPS comprised 6% of patients.(43)

The combination of doxorubicin and pembrolizumab was tested in a phase I/II trial exploring the concept of immune death induced by doxorubicin. This later was recommended at 75 mg/m² along with pembrolizumab. The response rate was distributed as partial response 22%, stable disease 59% and progressive disease 19%. The mPFS was 8.1 months (95% CI 6.3–10.8) being superior to historical controls considered by the authors 4.1 months (95% CI 3.0–6.6).(44)

Table 3 depicts outcomes of trials with anti-PD-1 alone or in combination in advanced STS. The combination of immune checkpoint inhibitors, especially with antiangiogenic agents, seemed to induce a clearly longer PFS in STS second line treatment, compared to anti-PD-1 alone, or anti-CTL4A alone or anti-angiogenic alone.(45)

Table 3:

List of trials conducted with anti PD-1 (in combination or in monotherapy in STS). Data of sunitinib in monotherapy in STS is also included for comparative purpose.

Study	Regimen	N	mPFS (m)	3-m PFS rate	6-m PFS rate	ORR (RECIST)	Included Subtypes	Responding Subtypes
Tawbi(37) (SARC028)	pembro	42	4.2	55%	32%	18% 7/40	UPS, LMS, LPS, SS	UPS, LPS, SS
D’Angelo(38) (A091401)	nivolumab	43	1.7	~ 35%	15%	5% 2/38	>10 (UPS, LMS, SS, LPS, ES…)	ASPS, LMS
Ben-Ami(75)	nivolumab	12	1.8	0%	0%	0%	uLMS	NA
George(45)	sunitinib	50	1.8	39%	22%	2% 1/48	Several: 23% LMS; 8% SS	DSRCT
Merchant(76)	ipilimumab	17	UNK	UNK	UNK	0% 0/17	Pediatric several SS, CCS, …	NA
D’Angelo(38) (A091401)	nivolumab- ipilimumab	42	4.1	~ 60%	28%	16% 6/38	>10 (UPS, LMS, SS, LPS, ES…)	LMS, UPS, Myxo, Angio
Wilky(40)	axitinib- pembro	33	4.7	65.6%	50%	25% 8/32	Several: 36% ASPS	ASPS, LMS, ES
Martin-Broto(43) (IMMUNOSARC)	nivolumab- sunitinib	50	5.9	69%	50%	11% 5/46	Several: 18% SS, 6% ASPS, UPS, ES…	ASPS, Angio, ECM, SS
Toulmonde(77)	metronomic cyclo-pembro	57	1.4	UNK	0% (LMS; UPS), 14.3%(Other STS)	2% 1/48	LMS, UPS, other STS, GIST	SFT
Pollack(44)	doxorubicin- pembro	37	8.1	UNK	UNK	22% 8/37	Several STS	UNK

UNK (unknown); NA (not applicable); Pembro (pembrolizumab); Cyclo (cyclophosphamide); ASPS (alveolar soft-part sarcoma); UPS (undifferentiated pleomorphic sarcoma); uLMS (uterine leiomyosarcoma); LMS (leiomyosarcoma); Angio (angiosarcoma); Myxo (myxofibrosarcoma); SS (synovial sarcoma); ECM (extraskeletal myxoid chondrosarcoma); LPS (liposarcoma); ES (epithelioid sarcoma).

Modified T-Cell Therapies for NY-ESO and MAGEA4

Cancer-testes antigens represent a family of antigens that arise from 276 genes (46). The most commonly studied within sarcomas are the New York Esophageal Tumor Antigen (NY-ESO) and the MAGEA4 antigen (MAGE Family Member A4), and more recently, the PRAME (Preferentially Expressed Antigen in Melanoma) (47,48). This review will focus on the most mature clinical data for synovial sarcoma, as the first demonstrated that NYESO is expressed in 80% of synovial sarcoma (49). His group went on to genetically engineer lymphocytes that were reactive to NY-ESO, and in a phase 1 clinical trial found on an objective tumor response in 4 of 6 patients with synovial sarcoma (49). More recently, a modified T-cell therapy called SPEAR (Specific Peptide Enhanced Affinity Receptor) T-cells was developed based on HLA:02 status and NY-ESO expression that was published as a phase 1 trial (50). Of the 42 patients treated, one patient had a complete response and 14 had a partial response. While the response rate was up to 50% in the cohort with high antigen expression and a conditioning regiment that included 30mg/m2 of fludarabine for 4 days and 1800mg/m2 of cyclophosphyamide for 2 days, with the response requiring a highly dose conditioning regiment. Of note, one case of aplastic anemia was seen with this regiment. This treatment is being further developed as a phase 2 clinical trial.

Most recently, a similar SPEAR T-cell targeting MAGEA4 in synovial sarcoma was presented at ESMO with an update at the connective tissue oncology (CTOS) meeting (51) (52). 7/14 patients demonstrated a partial response, and 6 of the 7 responders having durable responses to week 18 at the time of the data cut off with most patients still on trial. 13/14 patients had clinical benefit and the same 13/14 had at least grade 1 cytokine release syndrome in response to treatment. The durability of this target and therapy is awaiting and this is being formally testing in a Phase 2 registration trial. Finally, one patient on a high dose chemotherapy expansion cohort with 30mg/m2 of fludarabine for 4 days and 1800mg/m2 also developed aplastic anemia. What both patients here and above had in common were advanced age and extensive pretreatment. Therefore, clinical development utilizing high dose conditioning regiments in elderly heavily pretreated patients undergoing SPEAR T-cell therapies should be approached with cation.

Localized Immunotherapy approaches

Talimogene Laherparepvec (T-VEC) is an oncolytic immunotherapy based on intratumoral injection of a modified self-replicating human herpes virus type 1 that causes tumor lysis and antigen release. In a single center Phase 2 study, 20 patients were treated with T-VEC and pembrolizumab with the primary endpoint being objective response rate at 24 weeks. The ORR was 30% at 24 weeks, with the responding histologies being cutaneous angiosarcoma, UPS, myxofibrosarcoma, epithelioid sarcoma, and an unclassified sarcoma. There is need for further development of T-VEC in a randomized trial based on these results (53).

Hope within New Strategies of Immunotherapy for STS

T cells expressing MC.7.G5 TCR are able to kill a broad spectrum of tumor cell lines regardless of their HLA allomorph. This novel TCR acts through a protein called MR1 (major histocompatibility complex class I-related gene protein) and not through the largely described MHC, since anti-MR1, but not MHC I or MHC II antibodies blocked target-cell recognition by MC.7.G5. Of note, unlike MHC, MR1 sequence seems partially conserved amongst individuals, opening new doors for the development of novel pan-cancer, pan-population T cell–mediated cancer immunotherapy approaches.(54)

Moreover, other immune-checkpoint receptors, yet to be explored in a deeper way in sarcomas, such as LAG-3 or TIM-3, might also play an important role in this field. Therefore, following the recent immune-classification of STS microenvironment, mainly those belonging to the SIC E, expressed these immune-checkpoint proteins (LAG-3 and TIM-3).(55) The expression of LAG-3 has been correlated with worse overall survival in STS and its inhibition impaired tumor growth in immunocompetent 3-methylcholanthrene (MCA)-induced fibrosarcoma mouse models.(15) On the other hand, and in these immunocompetent mouse models, anti-TIM-3 antibodies were more effective in combination with anti-CTLA-4 or anti-PD-1 antibodies, in comparison to monotherapy.(56)

Bispecific antibodies are emerging as a new class of immunotherapeutic agents(57,58). At least 3 classes of bispecific antibodies are currently being developed: i) cytotoxic effector cell redirectors; ii) tumor-targeted immunomodulators and iii) dual immunomodulators. The cytotoxic effector cell redirectors engage a tumor-associated antigen with the complex CD3 T-cell co-receptor, thus guiding T-cell cytotoxic effect directly towards the tumor cells. As an example, Orlotamab is a T-cell engager that targets both B7-H3 and CD3. Noteworthy, high expression of B7-H3 gene (CD276) has been described in STS, more precisely in dedifferentiated liposarcoma, undifferentiated pleomorphic sarcoma and myxofibrosarcoma.(4) Amongst the tumor-targeted immunomodulators, special interest should be reserved for tumor-targeting 4–1BB agonist, composed by a trimeric 4–1BB ligand, a Fab moiety targeting stromal fibroblast activation protein (FAP), and a silenced Fc domain that lacks affinity for C1q and FcγRs (59,60), since FAP seems to be consistently expressed in STS(61,62). Moreover, simultaneously targeting two immune-checkpoints is a promising concept that can be achieved with dual immunomodulators. Several dual immunomodulators are being developed, targeting PD-1 and LAG-3 (e.g. MGD013 or FS118), PD-1 and TIM-3 (e.g. MCLA-134) or PD-1 and CTL-4 (e.g. XmAb20717). There efficacy in STS awaits formal testing.

Conclusions

In summary, we are still at the beginning of the beginning of our understanding of immunotherapy in sarcoma. From 1891 to the present, individual patients have been rendered disease free using immunotherapy approaches. Overall, combination regimens, based on immune checkpoint inhibitors, seemed to be more efficient compared to monotherapy (e.g. anti-PD-1 or anti-CTL4); specially the combination of anti-PD-1 with anti-angiogenic agents. Moreover, modified T-Cell therapies are currently being tested in specific STS subtypes with a significant clinical benefit for the patients. However, further studies are needed to describe novel antigens in other STS subtypes. Likewise, new therapeutic strategies, such as dual immunomodulators, deserves to be tested in the context of STS. It will take the combined partnership between the international sarcoma centers, patients and basic scientists to fully understand and optimize the use of immunotherapy for STS.