Cell Liposarcoma Research Articles

Abstract 2954: Chromatin accessibility dynamics in CDK4/6 inhibitor-induced senescence

Abstract Liposarcomas are a rare cancer that often undergo amplification of the 12q13-15 locus, which includes the gene encoding the cyclin-dependent kinase CDK4; CDK4/6 inhibitors therefore offer a promising targeted therapy to halt proliferation in these tumors where therapeutic options are scarce once surgical resection is no longer possible. CDK4/6 inhibition can lead to either reversible or irreversible exit from the cell cycle (quiescence or senescence, respectively), but the underlying mechanisms are not well understood. Applying ATAC-seq in a liposarcoma cell line model of therapy-induced senescence, we have assayed alterations in the chromatin landscape that arise in response to CDK4/6 inhibitor treatment to identify epigenetic changes that drive this cell fate decision. Additionally, we have compared this with cells that undergo senescence due to DNA damage induced by the drug doxorubicin, an anthracycline that is the standard first-line chemotherapy for liposarcoma. In CDK4/6 inhibition, we identified dynamic patterns of accessibility as cells transition from quiescence to senescence over 28 days of treatment. NF-kB motifs in promoter-proximal regions displayed increased accessibility as cells commit to the senescence state, and ATAC-seq peaks with AP-1 transcription factor family motifs exhibit dynamic accessibility. We characterized the DNA damage response by gamma-H2AX and 53BP1 immunofluorescence and found that for CDK4/6 inhibition there is minimal DNA damage and only at late senescence time points, in marked contrast with doxorubicin treatment. We observed differences in accessibility between CDK4/6 inhibitor- and doxorubicin-induced senescence in a region on chromosome 6 that contains the gene FRK. This gene encodes a tyrosine kinase in the Src family that negatively regulates cell proliferation and has been implicated as a tumor suppressor through upregulation of p21, which may reinforce restriction of phosphorylation on Rb in parallel with CDK4/6 inhibition. These findings provide further evidence of the epigenetic impact of CDK4/6 inhibitors in cancer cells, and highlight shared and specific mechanisms that lead to therapy-induced senescence. Citation Format: Justin Rendleman, Joanna Yeung, Nicole Pagane, Irene Duba, Andrew Scortea, Andrew Koff, Viviana I. Risca. Chromatin accessibility dynamics in CDK4/6 inhibitor-induced senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2954.

ZENYTH-ESO: Master protocol to assess the safety and recommended phase II dose of next generation NY-ESO-1-specific TCR T-cells in HLA-A*02 patients with synovial sarcoma and myxoid/round cell liposarcoma [Substudy 3, GSK4427296

TPS2681 Background: Letetresgene autoleucel (lete-cel; GSK3377794) is an autologous T-cell therapy expressing an affinity-enhanced T-cell receptor (TCR) to improve recognition of cancer cells expressing NY-ESO-1 and/or LAGE-1a. Next generation NY-ESO-1 TCR T-cell therapy, GSK4427296, utilizes the same TCR as lete-cel, as well as an epigenetic reprogramming process (Epi-R) developed by Lyell Immunopharma to alter the phenotypic T-cell profile of the manufactured product, and is intended to increase the proportion of cells with properties of durable stemness. T cells with properties of durable stemness are able to proliferate, persist, and self-renew with anti-tumor functionality. A first-time-in-human master protocol (NCT04526509) is underway to evaluate the safety, tolerability, and recommended phase II dose (RP2D) of next generation NY-ESO-1 TCR T-cell therapies. Substudy 3 is added to this master protocol to assess GSK4427296 in patients with advanced synovial sarcoma (SS) or myxoid/round cell liposarcoma (MRCLS). Methods: This substudy includes a dose confirmation stage to assess RP2D and a dose expansion stage, aiming to dose 10 participants at the RP2D. Key inclusion criteria: age ≥18 years; measurable disease per RECIST v1.1; HLA-A*02:01, A*02:05, or A*02:06 positivity; NY-ESO-1/LAGE-1a tumor expression; advanced (metastatic/unresectable) SS with t(X;18) translocation or MRCLS with a translocation involving DDIT3 and/or FUS and/or EWSR1 genes; and anthracycline-based therapy receipt/completion/intolerance. Key exclusion criteria: prior malignancy that is not in complete remission or clinically significant systemic illness; prior receipt of gene or allogenic stem cell/solid organ transplant; and central nervous system metastases. Primary endpoints: safety (adverse events) and tolerability (dose-limiting toxicities). Secondary endpoints: investigator-assessed overall response rate, duration of response, maximum transgene expansion (Cmax), Tmax, and AUC(0-t). Analyses will be descriptive. The master protocol is open for recruitment. Clinical trial information: NCT04526509.

Identification of response stratification factors from pooled efficacy analyses of afamitresgene autoleucel (“Afami-cel” [Formerly ADP-A2M4]) in metastatic synovial sarcoma and myxoid/round cell liposarcoma phase 1 and phase 2 trials.

11562 Background: Afami-cel is an autologous, HLA-A*02-restricted, specific peptide enhanced affinity receptor, T-cell therapy engineered to target MAGE-A4+ solid tumors. The pivotal, 2-cohort, single-arm, Phase 2, SPEARHEAD-1 trial (NCT04044768) with afami-cel met its primary endpoint based on Cohort 1 data. As of September 1, 2021, in 47 patients (pts) with metastatic synovial sarcoma (SyS) or myxoid/round cell liposarcoma (MRCLS), the overall response rate (ORR) per independent review was 34% with encouraging durability (Van Tine, et al. Paper 30: CTOS 2021; Virtual). To identify potential stratification factors for response and assess whether response is a proxy for progression-free survival (PFS), we present pooled analyses using data from the prior Phase 1 trial (NCT03132922) and Cohort 1 of the SPEARHEAD-1 trial. Methods: Eligible pts (16–75 years) were HLA-A*02+ with MAGE-A4+ tumors. Pts received afami-cel after lymphodepleting chemotherapy. The pooled analyses evaluated ORR per RECIST v1.1 by investigator review, stratified by 7 factors, and safety. Results: In the pooled data, 69 pts received afami-cel (2.12–9.99×109 transduced T-cells) and were evaluable for response (Phase 1, n = 18; Phase 2, n = 51); all expressed one eligible HLA-A*02 allele. Median (range) for: age was 42 years (19–76), number of prior lines of therapy was 2 (1–12), and tumor MAGE-A4 H-score was 230 (60–300). Median (range) H-score was higher in SyS (256 [60–300]) than in MRCLS (180 [112–230]). The pooled investigator-assessed ORR was 36.2% (40.7% in SyS; 10.0% in MRCLS). Responses occurred across a wide MAGE-A4 H-score range (134–300). Median (range) duration of response was 52 weeks (8.29–75.14). Response rate was higher in the 59 pts with SyS: with ≤2 vs ≥3 prior lines of therapy (55.2% vs 26.7%), baseline target lesion sum of longest diameters <10cm vs ≥10cm (53.1% vs 25.9%), MAGE-A4 H-score ≥200 vs <200 (46.3% vs 27.8%), without vs with bridging therapy (48.6% vs 29.2%), who were female vs male (46.4% vs 35.5%), aged ≥40 vs <40 years (45.7% vs 33.3%), and from North America vs Europe (42.6% vs 33.3%). In responders vs non-responders with SyS, respectively, median PFS was 58.3 vs 11. 0 weeks (log-rank p-value <0.0001); the probability of being progression-free at 24 weeks was 0.8 vs 0.2. The pooled benefit:risk profile of afami-cel was similar to that in the SPEARHEAD-1 trial (Van Tine, et al. Paper 30: CTOS 2021; Virtual.). Conclusions: We show that baseline tumor burden, prior systemic treatment history, and MAGE-A4 tumor expression levels are potential factors associated with response to afami-cel, although their true predictive value for response status awaits confirmation. Our findings will inform the ongoing clinical development of afami-cel in sarcoma, especially for prognostic studies with PFS or overall survival endpoints. Clinical trial information: NCT04044768, NCT03132922.

Primary efficacy and safety of letetresgene autoleucel (lete-cel; GSK3377794) pilot study in patients with advanced and metastatic myxoid/round cell liposarcoma (MRCLS).

11500 Background: Lete-cel is an autologous T-cell therapy targeting NY-ESO-1 tumors using a genetically modified, high-affinity T-cell receptor. MRCLS is a sarcoma with poor response to current immunotherapy approaches and limited treatment options. The cancer testis antigen NY-ESO-1 is expressed in 80‒90% of MRCLS tumors, making this a promising target. This report summaries the primary efficacy and safety results of a pilot study of lete-cel in patients (pts) with advanced or metastatic MRCLS. Methods: This is an open label, study of lete-cel in pts with advanced or metastatic MRCLS following reduced-dose (Cohort 1 [C1]; 30 mg/m2 fludarabine [flu] x 3d + 600 mg/m2 cyclophosphamide [cy] x 3d) or standard dose (Cohort 2 [C2]; 30 mg/m2 flu x 4d + 900 mg/m2 cy x 3d) lymphodepletion (LD). Key eligibility criteria were: age ≥18 y; HLA-A*02:01; A*02:05, or A*02:06; advanced or metastatic NY-ESO-1+ MRCLS (≥30% of cells 2+/3+ by IHC); prior anthracycline treatment, and measurable disease. The transduced T cell dose range was 1– 8 × 109. Response was assessed at weeks 4, 8, 12, 24, then every 3 months (mo) until disease progression, death, or withdrawal. Investigator-assessed (IA) ORR by RECIST v1.1 was the primary efficacy endpoint. Secondary endpoints included safety, independently assessed ORR by RECIST v1.1, time to response (TTR), duration of response (DOR), progression-free survival (PFS). Overall survival (OS) was an exploratory endpoint. Results: 23 pts enrolled from March 2017 to February 2020. The median age was 47.0 yrs (range 33 to 72). 20 pts were dosed with T cells, 10 in each cohort with a median transduced T cell dose of 4.6 x 109. 8 of 20 pts (40%) had 1 line of prior therapy, 6 pts (30%) had 2 lines, and 6 pts (30%) had ≥3 lines. The median follow-up was 5.6 (C1) and 12.9 (C2) mo. In C1 the IA ORR was 20%, with best response (BR) of partial response (PR) in 2 pts and BR of stable disease (SD) in 8 pts. The median TTR was 1.9 mo, median DOR was 5.3 mo (95% CI: 1.9-8.7), and median PFS was 5.4 mo (95% CI: 2.0-11.5). In C2 the IA ORR was 40%, with BR of PR in 4 pts and BR of SD in 5 pts. The median TTR was 1.9 mo, median DOR was 7.5 mo (95% CI: 6.0-NE), and median PFS was 8.7mo (95% CI: 0.9-NE). OS is not yet mature. All pts experienced at least 1 treatment-emergent adverse event (TEAE). 55% of pts experienced serious TEAEs. 90% of pts had Gr ≥3 TE neutropenia, with 83% probability of resolution of initial Gr ≥3 occurrence by Day 30. Cytokine release syndrome occurred in 80% of pts, of which 25% were Gr 3, with 1st onset within 5d of infusion and median duration 7.5d. No Graft-vs-host disease, immune effector cell–associated neurotoxicity syndrome, Guillain-Barré Syndrome were reported. Conclusions: Treatment with a single dose of lete-cel showed anti-tumor activity, including response and long median PFS with an acceptable safety profile in pts with advanced and metastatic MRCLS. Clinical trial information: NCT02992743.

A phase 1/2 dose escalation/expansion study of OR2805 alone or in combination in subjects with advanced solid tumors.

TPS2693 Background: M2-like macrophages are thought to promote an immunoinhibitory environment and prevent activation and proliferation of T cells, leading to resistance to checkpoint inhibitor (CPI) therapy. OR2805 is a fully human monoclonal antibody identified in a patient who had an exceptional response to CPI. OR2805 recognizes CD163, an immunosuppressive scavenger receptor highly expressed on M2 tumor-associated macrophages (TAMs). OR2805 treatment reprograms M2 macrophages to take on an immunostimulatory, M1-like phenotype. In macrophage/T cell coculture assays, OR2805 treatment relieved the suppressive effect of M2c macrophages as demonstrated by increased T-cell proliferation and stimulation of IFN-γ and perforin release. OR2805 restored the IFN-γ production of exhausted T cells and showed a synergistic effect on cocultures treated in combination with CPI. OR2805 monotherapy demonstrated significant anti-tumor activity in lung cancer xenograft models in NSG-SGM3 mice. Relieving the immune suppression of CD163-expressing TAMs by OR2805 to improve anti-tumor T-cell responses will be evaluated alone and in combination with CPI therapy. Methods: This phase 1/2, open-label, multicenter, dose-escalation/expansion study is assessing the safety, tolerability and preliminary activity of OR2805 alone and in combination with anti-PD-1 or chemotherapy in up to 325 adults (≥18 years) with histologically or cytologically confirmed metastatic or locally advanced solid tumors. The study is divided into 3 parts. Part A will enroll subjects with advanced solid tumors of any histology to describe the preliminary safety profile of OR2805 and determine the recommended phase 2 dose (RP2D) of OR2805 alone and in combination with an anti-PD-1 monoclonal antibody. Part B will further characterize the safety and preliminary antitumor activity of OR2805 either alone or in combination with an anti-PD-1 or chemotherapy in previously treated non-small cell lung cancer (NSCLC), melanoma and liposarcoma, as well as in previously untreated NSCLC in combination with an anti-PD-1 antibody. Part C will evaluate OR2805 monotherapy in relapsed squamous cell carcinoma of the head and neck and other tumor types with an emphasis on correlative biomarker analysis. OR2805 will be infused on Day 1 of 21-day cycles. Primary endpoints are adverse events, laboratory abnormalities, dose-limiting toxicities, and dose-level safety and activity. Secondary endpoints are objective response rates, duration of response, complete response, progression-free survival, overall survival, PK, and antidrug antibodies. Exploratory biomarkers of OR2805-mediated pharmacodynamic effects and immune stimulation will be explored. The study was opened September 2021 and is enrolling at sites in the United States with planned expansion within the US and to Europe. Clinical trial information: Clinical trial information: NCT05094804.

Establishment and characterization of two novel patient-derived myxoid liposarcoma cell lines.

Myxoid liposarcoma (MLPS) is a lipogenic sarcoma, characterized by myxoid appearance histology and the presence of the FUS-DDIT3 fusion gene. MLPS shows frequent recurrence and poor prognosis after standard treatments, such as surgery. Therefore, novel therapeutic approaches for MLPS are needed. Development of novel treatments requires patient-derived cell lines to study the drug responses and their molecular backgrounds. Presently, only three cell lines of MLPS have been reported, and no line is available from public cell banks. Thus, this study aimed to establish and characterize novel MLPS cell lines. Using surgically resected tumor tissue from two patients with MLPS, two novel lines NCC-MLPS2-C1 and NCC-MLPS3-C1 were established. The presence of FUS-DDIT3 fusion, slow growth, spheroid formation, and invasive capability in these cell lines was confirmed. Growth retardation was monitored for 213 anti-cancer agents using NCC-MLPS2-C1 and NCC-MLPS3-C1 cells, and the results were integrated with the response to treatments in an MLPS cell line, NCC-MLPS1-C1, which was previously established in our laboratory. We found that romidepsin suppressed cell proliferation at considerably low concentrations in all three examined cell lines. NCC-MLPS2-C1 and NCC-MLPS3-C1 cell lines developed here represent a useful tool for basic and preclinical studies of MLPS.

Heterogeneous Skin Phantoms for Experimental Validation of Microwave-Based Diagnostic Tools.

Considerable exploration has been done in recent years to exploit the reported inherent dielectric contrast between healthy and malignant tissues for a range of medical applications. In particular, microwave technologies have been investigated towards new diagnostic medical tools. To assess the performance and detection capabilities of such systems, tissue-mimicking phantoms are designed for controlled laboratory experiments. We here report phantoms developed to dielectrically represent malign skin lesions such as liposarcoma and nonsyndromic multiple basal cell carcinoma. Further, in order to provide a range of anatomically realistic scenarios, and provide meaningful comparison between different phantoms, cancer-mimicking lesions are inserted into two different types of skin phantoms with varying tumor–skin geometries. These configurations were measured with a microwave dielectric probe (0.5–26.5 GHz), yielding insight into factors that could affect the performance of diagnostic and detection tools.

When is a lipoma not a lipoma? Case report presenting a lipoblastoma-like tumor of the gluteal cleft in an older gentleman with literature review.

Introduction and importanceLipoblastoma-like tumors are rare tumors that can be confused with lipoblastomas and liposarcomas but have distinct characteristics. This tumor has previously been identified in the vulva of females, and recently in isolated cases of young males. Given its rarity, we present an instance of this tumor in an older man, demonstrating that this pathology is not limited to a specific age or sex, and surgeons and pathologists must be aware of it in their differential.Case presentationA 58-year-old male presented for evaluation of an enlarging mass in his right gluteal cleft. Prior to referral for surgical evaluation, the patient underwent an ultrasound-guided biopsy of the mass. Histologically, the tumor was a low-grade cellular spindle cell neoplasm in a fibrous to myxoid stroma. Immunohistochemical and molecular workup ruled out several malignant mesenchymal neoplasms, including myxoid liposarcoma, dedifferentiated liposarcoma, melanoma, low-grade fibromyxoid sarcoma, and sarcomatoid carcinoma. The patient initially declined surgery, but the mass continued to grow, and excision was chosen given the uncertain pathology. The tumor was resected with negative margins and histologically characterized as a “lipoblastoma-like lesion”, with features of a myxoid liposarcoma and spindle cell lipoma. Seven months post-resection, there were no signs of recurrence or metastasis.Clinical discussionDespite radiologic and pathologic similarities to malignant lipomatous tumors, lipoblastoma-like tumors are benign and have a good prognosis.ConclusionsClinicians should be aware of this entity despite its rarity as resection with negative margins is curative and may be needed to rule out more aggressive tumors.

A mesenchymal chondrosarcoma with aberrant nuclear expression of STAT6: a potential diagnostic pitfall

STAT6 is usually considered to be a very sensitive and specific immunomarker for diagnosis of solitary fibrous tumor (SFT), being a surrogate of the NAB2-STAT6 fusion gene identified in most cases of this tumor. STAT6 expression has also been reported in rare cases of other soft tissue tumors, such as low-grade fibromyxoid sarcoma, myxoid/round cell liposarcoma, dedifferentiated liposarcoma and deep fibrous histiocytoma. The aim of this study was to report, for the first time, a case of mesenchymal chondrosarcoma showing diffuse aberrant immunohistochemical expression of STAT6. Molecular biology, showing the HEY1-NCOA2 fusion gene, was crucial to rule out SFT.

Improving Immunotherapy Efficacy in Soft-Tissue Sarcomas: A Biomarker Driven and Histotype Tailored Review.

Anti-PD-(L)1 therapies yield a disappointing response rate of 15% across soft-tissue sarcomas, even if some subtypes benefit more than others. The proportions of TAMs and TILs in their tumor microenvironment are variable, and this heterogeneity correlates to histotype. Tumors with a richer CD8+ T cell, M1 macrophage, and CD20+ cells infiltrate have a better prognosis than those infiltrated by M0/M2 macrophages and a high immune checkpoint protein expression. PD-L1 and CD8+ infiltrate seem correlated to response to immune checkpoint inhibitors (ICI), but tertiary lymphoid structures have the best predictive value and have been validated prospectively. Trials for combination therapies are ongoing and focus on the association of ICI with chemotherapy, achieving encouraging results especially with pembrolizumab and doxorubicin at an early stage, or ICI with antiangiogenics. A synergy with oncolytic viruses is seen and intratumoral talimogene laherpavec yields an impressive 35% ORR when associated to pembrolizumab. Adoptive cellular therapies are also of great interest in tumors with a high expression of cancer-testis antigens (CTA), such as synovial sarcomas or myxoid round cell liposarcomas with an ORR ranging from 20 to 50%. It seems crucial to adapt the design of clinical trials to histology. Leiomyosarcomas are characterized by complex genomics but are poorly infiltrated by immune cells and do not benefit from ICI. They should be tested with PIK3CA/AKT inhibition, IDO blockade, or treatments aiming at increasing antigenicity (radiotherapy, PARP inhibitors). DDLPS are more infiltrated and have higher PD-L1 expression, but responses to ICI remain variable across clinical studies. Combinations with MDM2 antagonists or CDK4/6 inhibitors may improve responses for DDLPS. UPS harbor the highest copy number alterations (CNA) and mutation rates, with a rich immune infiltrate containing TLS. They have a promising 15-40% ORR to ICI. Trials for ICB should focus on immune-high UPS. Association of ICI with FGFR inhibitors warrants further exploration in the immune-low group of UPS. Finally translocation-related sarcomas are heterogeneous, and although synovial sarcomas a poorly infiltrated and have a poor response rate to ICI, ASPS largely benefit from ICB monotherapy or its association with antiangiogenics agents. Targeting specific neoantigens through vaccine or adoptive cellular therapies is probably the most promising approach in synovial sarcomas.

Abstract P087: Targeting LSD1 protein scaffolding function in FET-rearranged sarcomas with SP-2577

Abstract The RNA-binding proteins, FUS, EWSR1, and TAF15 (FET protein family) are frequently fused to transcription factors, and the resulting oncogenic fusion proteins characterize various sarcomas. These fusion proteins act as aberrant transcription factors and promote oncogenic gene expression profiles. FET family rearranged sarcomas include Ewing Sarcoma, desmoplastic round cell tumors, myxoid liposarcoma, and clear cell sarcoma, amongst others. A growing body of work suggests that, independently of Lysine Specific Demethylase 1 (LSD1) demethylase activity, LSD1’s association with FET family oncogenic transcription factors is critical for FET-rearranged sarcoma development and progression. Extensive ongoing work aims to characterize the functional relationship of LSD1 and EWS/FLI, as well as the anti-tumor effect of disrupting that interaction. SP-2577 is an oral, first-in-class, small molecule with reversible, noncompetitive inhibition of LSD1 demethylase activity (IC50: 25–50 nM) and, importantly, also disrupts LSD1 protein-protein interactions. In vitro and in vivo data demonstrate SP-2577, or analogs, reverses EWS/ETS-mediated transcriptional regulation at both up- and downregulation target genes, leading to significant tumor growth inhibition in Ewing Sarcoma mouse xenograft studies. In this study, we set out to expand our understanding of SP-2577’s efficacy in diverse FET-rearranged malignancies including desmoplastic small round cell tumor (DSCRT: EWSR1/WT1), myxoid liposarcoma (ML: FUS/DDIT3), and clear cell sarcoma (CCS: EWS/ATF1). To examine whether these additional FET-rearranged sarcomas are sensitive to LSD1 scaffolding inhibition, we measured viability of Ewing sarcoma cells A673, TC32, TTC-446, and SK-N-MC, clear cell sarcoma cell lines SU-CCS-1, DTC-1, myxoid liposarcoma cell lines DL-221, 402-91, and 1765-92, and DSCRT patient derived xenograft organoids from SJDSRCT046151, X0069, SJDSRCT046155 after 96 hours of treatment with the clinical compound SP-2577, a first generation compound SP-2509, an inactive control, SP2513, and, a TCP-scaffold based, irreversible inhibitor of LSD1, OG-L002. While SP-2513 and OG-L002 had no activity against these cells SP-2509 and SP-2577 demonstrated potent activity across cell lines in vitro. Preliminary in vivo assessment of SP-2577 efficacy in DSCRT PDX SJDSRCT046151 resulted in a significant delay in time to event (p<0.02). Together, these data demonstrate that the unique scaffolding inhibition function of SP-2577 is essential for reducing cell viability through LSD1 targeting. Currently, we are elucidating changes to the transcriptome and proteome following SP-2577 treatment, in vitro. These data, in combination with results from the ongoing dose expansion phase 1 trial of SP-2577 in select sarcoma patients, may offer further mechanistic insight into the dependency of FET-rearranged sarcomas on LSD1 scaffolding functions and potentially lead to identifying biomarkers of sensitivity and response. Citation Format: Galen Rask, Emily R. Theisen, Aundrietta D. Duncan, Daniela Y. Santiesteban. Targeting LSD1 protein scaffolding function in FET-rearranged sarcomas with SP-2577 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P087.

Anti-Tumor Effect of Apatinib and Relevant Mechanisms in Liposarcoma.

BackgroundPrimary retroperitoneal liposarcomas (RLPSs) are rare heterogeneous tumors for which there are few effective therapies. Certain anti-angiogenic tyrosine kinase inhibitors have demonstrated efficacy against various solid tumors. The aims of this study were to investigate the effect of Apatinib against retroperitoneal liposarcoma cells and its underlying mechanism and to explore the anti-tumor efficacy of a combination of Apatinib and Epirubicin.MethodsCD34 immunohistochemical staining was used to measure microvessel density (MVD) in 89 retroperitoneal liposarcoma tissues. We used CCK-8 cell proliferation, clone formation, Transwell migration, invasion assays and flow cytometry to evaluate the effects of Apatinib alone and the combination of Apatinib and Epirubicin on liposarcoma cells. High-throughput RNA sequencing and western-blotting was used to identify key differentially expressed genes (DEGs) in SW872 cell line after application of Apatinib. Murine patient-derived tumor xenograft (PDX) was established to assess the efficacy and safety of Apatinib monotherapy and the combination of Apatinib and Epirubicin in RLPS.ResultsThe microvessel density (MVD) varied widely among retroperitoneal liposarcoma tissues. Compared with the low-MVD group, the high-MVD group had poorer overall survival. Apatinib inhibited the liposarcoma cell proliferation, invasion and migration, increased the proportion of apoptosis, and induced G1 phase arrest. In addition, the combination of Apatinib and Epirubicin enhanced the foregoing inhibitory effects. High-throughput RNA sequencing showed that Apatinib downregulated the expression of TYMS and RRM2. Western blotting verified that Apatinib downregulated the TYMS/STAT3/PD-L1 pathway and inhibited liposarcoma proliferation by suppressing the RRM2/PI3K/AKT/mTOR pathway. In the murine PDX model of retroperitoneal liposarcoma, Apatinib and its combination with Epirubicin significantly inhibited microvessel formation and repressed tumor growth safely and effectively.ConclusionsApatinib and its combination with Epirubicin showed strong efficacy against liposarcoma both in vitro and in vivo. Apatinib might inhibit liposarcoma cell proliferation through the RRM2/PI3K/AKT/mTOR signaling pathway and downregulate PD-L1 via the TYMS/STAT3 signaling pathway.

Role of FUS-CHOP in Myxoid Liposarcoma via miR-486/CDK4 Axis.

This study aimed to explore the roles and relationship between FUsed in Sarcoma (FUS)-C/EBP HOmologous Protein (CHOP), microRNA (miR)-486 and cyclin dependent kinase 4 (CDK4) in myxoid liposarcoma, and determined whether FUS-CHOP can regulate proliferation and apoptosis of myxoid liposarcoma cells by regulating miR-486/CDK4 axis. The levels of miR-486, CDK4 and FUS-CHOP in myxoid liposarcoma samples/adjacent normal muscle tissues and myxoid liposarcoma/human adipose-derived stem cell line were evaluated using reverse transcription-quantitative polymerase chain reaction and western blotting. Cell proliferation and apoptosis were performed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide and flow cytometry, respectively. Furthermore, the apoptosis-related proteins were determined using Western blot assay. We found that miR-486 was down-regulated, FUS-CHOP and CDK4 were up-regulated in myxoid liposarcoma tissues and myxoid liposarcoma cell lines. Moreover, FUS-CHOP-siRNA distinctly suppressed FUS-CHOP level and increased miR-486 levels in 1955/91 cells. Our results demonstrated that knockdown of FUS-CHOP by siRNA inhibited 1955/91 growth, promoted cell apoptosis and enhanced cleaved Caspase3 protein expression. However, all these data were reversed by miR-486 inhibitor. Similarly, compared to mimic control, miR-486 mimic markedly reduced 1955/91 cells growth, induced cell apoptosis and fortified cleaved Caspase3 level, while these results were abolished by CDK4-plasmid. Collectively, our observations clearly suggested that FUS-CHOP regulated myxoid liposarcoma cell proliferation and apoptosis by the regulation of miR-486/CDK4 axis, indicating the potential use of FUS-CHOP-siRNA as a promising therapy for myxoid liposarcoma.

391 Biomarker correlates of response in patients with advanced myxoid/round cell liposarcoma (MRCLS) treated with NY-ESO-1 TCR T cells (Letetresgene autoleucel)

BackgroundThis is an open label pilot study (NCT02992743) on letetresgene autoleucel (lete-cel; GSK3377794), an NY-ESO-1-specific autologous CD4+ and CD8+ T cells expressing a high affinity T-cell receptor which recognizes the NY-ESO-1 antigen epitope in complex with specific HLA- alleles A*02, which exhibited anti-tumor activity and manageable safety profiles in patients with advanced MRCLS based on interim analysis (IA) data.1 Lymphodepletion has been shown to enhance the expansion, persistence, and homing of therapeutically infused T-cells, thereby potentiating therapeutic efficacy against malignant diseases.2 Initial T-cell kinetics data from this study demonstrated that lymphodepletion regimen (LDR)-B robustly depleted lymphocytes at infusion and was trended with higher peak cell expansion (Cmax) vs. LDR-A. The peak expansion was significantly associated with weight-normalized transduced cell dose and trended with response.1 Here, we will be discussing additional cell kinetics data and other exploratory biomarker correlates of response.MethodsPatients with advanced MRCLS were enrolled to 2 cohorts and received either planned A (N=10) or B (N=10) LDRs prior to lete-cel infusion (table 1). Response was assessed per RECIST v1.1. Transduced cell kinetics were measured by quantitative PCR of transgene vector copies in DNA from peripheral blood mononuclear cells (PBMCs). Serum cytokines (Meso Scale Discovery immunoassay) as pharmacodynamic (PD) markers of response and their association with T cell kinetics will be discussed. Phenotypic characterization of the cell product (pre- and post- infusion) via Flow cytometry using Cytek Aurora (23 color panel), to help understand correlation of response with engineered cell product attributes, will be presented. Potential biomarker correlates of clinical response were tested using generalized linear models.ResultsFive out of 6 responders with available lab data exhibited robust lymphocyte depletion at infusion (0–25 cell/µL) and high Cmax (>50,000 vector copies/µg gDNA) with LDR. Only 6/14 non-responders exhibited low lymphocytes counts at infusion and high Cmax. LDR-B also induced strong depletion of monocytes at infusion (p=0.03) vs. LDR-A, but depletion of monocytes did not show association with response. Higher Cmax was correlated with exposure (AUC0–28d) (Adj. R2=0.606). AUC0–28d was a better predictor of response in patients receiving LDR-B (p=0.0182), with AUC0–28d trending towards predicting response in the LDR-A cohort. AUC0–28d was associated with tumor volume reduction (p=0.0569).Abstract 391 Table 1ConclusionsExposure–response analysis of this study reveals that efficacy appears to be driven by weight-normalized transduced cell dose and LDR via AUC0–28d. Higher AUC0–28d was correlated with Cmax and maximum tumor volume reduction.AcknowledgementsThis study (208469; NCT02992743) was funded by GlaxoSmithKline.Trial RegistrationNCT02992743ReferencesD’Angelo SP, et al. J Clin Oncol 2021;39:15_suppl:11521.Bechman, Maher. Expert Opin Biol Ther 2021;21(5):627–637.Ethics ApprovalThis study was approved by institutional review boards (IRB) at the six participating sites.

The Role of Ifosfamide-Doxorubicin Chemotherapy in Histology-Specific, High Grade, Locally Advanced Soft Tissue Sarcoma, Treated With Wide Excision Plus Radiation

<h3>Purpose/Objective(s)</h3> To compare long-term outcomes of high grade primary soft tissue sarcoma (STS), using ifosfamide-doxorubicin vs local therapy alone, in histology-specific sarcomas. <h3>Materials/Methods</h3> Retrospective analysis was performed on 127 patients from 2005 to 2018, who had high grade STS of extremity or trunk, > 5cm, that were either Synovial Cell, Dedifferentiated Liposarcoma (DDL), Myxofibrosarcoma, Round Cell Liposarcoma (RCLS), Undifferentiated Pleomorphic Sarcoma (UPS), or Undifferentiated Sarcoma not otherwise specified (NOS), with central pathology review. Ifosfamide-doxorubicin was given over 4-5 cycles, with chemotherapy given in 38 patients, while 89 received local therapy alone. Pre-operative radiation was given in 93 (73.2%), postoperative in 31 (24.4%), and amputation in 3 (2.4%). Multi-variable analysis of prognostic factors was performed, and local-recurrence-free-survival (LRFS), distant-metastases-free-survival (DMFS), disease-specific-survival (DSS), and overall-survival (OS) were estimated using Kaplan-Meier. <h3>Results</h3> Median follow-up was 4.5 years. Younger age (<i>P</i> < 0.0001) and Synovial histology (<i>P</i> = 0.0002) were more likely to undergo chemotherapy. Size > 10cm and trunk location were poor prognostic features on multivariable analysis (MVA) affecting DMFS, DSS, and OS, while DDL had more favorable survival; although size, trunk location, and DDL histology were not significantly different between treatment groups. Sequence of radiation did not significantly affect any of the survival outcomes. Ifosfamide-doxorubicin vs local therapy alone improved 5-year DMFS at 70.2% vs 49.5%, <i>P</i> = 0.02, DSS 83.5% vs 57.9%, <i>P</i> = 0.009, and OS 80.6% vs 53.8%, <i>P</i> = 0.002. Sub-analysis of non-synovial histology still showed a significant improvement in favor of chemotherapy in DMFS, (<i>P</i> = 0.04), DSS (<i>P</i> = 0.02), and OS (<i>P</i> = 0.003). <h3>Conclusion</h3> Ifosfamide-doxorubicin benefits younger patients with > 5cm, high grade, STS of the trunk or extremity in Synovial Cell, DDL, Myxofibrosarcoma, RCLS, UPS, and Undifferentiated Sarcoma NOS.

Abstract 1067: PDK-1 targeting with SNS-510 in sarcoma

Abstract Soft tissue sarcomas are heterogeneous mesenchymal neoplasms and account for 1% of all cancers in adults. Over 50 sarcomas subtypes have been classified and many have limited treatment options some of which include surgery, chemotherapy and radiation. 3-phosphoinositide dependent protein kinase 1 (PDK1) is overexpressed in many cancers, suggesting a role for PDK1 in cancer progression. In addition, PDK1 has been identified as a key modifier of acquired resistance to CDK4/6 inhibitors. In order to interrogate this in sarcoma, we elected to test SNS-510 a potent, orally bioavailable PDK1 inhibitor against a broad range of sarcoma cell lines (leiomyosarcoma, dedifferentiated liposarcoma, Ewing's sarcoma, osteosarcoma, chondrosarcoma and synovial sarcoma) as well as dedifferentiated liposarcoma cell lines with acquired resistance to ribociclib, which are also cross resistant to palbociclib. All the cell lines expressed PDK1 and, as measured by WST-8 cell proliferation assay, nearly all the cell lines were sensitive to the drug, with IC50s generally between 250 to 500nM. Leiomyosarcoma cell lines (SK-LMS, SK-UT1, SK-UT1b) were seen to be most sensitive to SNS-510 with IC50s determined to be between 50-100nM. In contrast, synovial sarcoma cell lines (SYO-1 and HSSY-II) exhibited relative resistance to SNS-510 with IC50s determined to be &amp;gt; 500nM. As assessed by western blot, SNS-510 exhibited a dose dependent inhibition of the PDK-1/RSK2/AKT pathway with inhibition of p-RSK2(244), p-AKT(308), and pS6(235/236). SNS-510 induced a dose-dependent G1 cell cycle arrest. This was associated with inhibition of p-RB(780) and suppression of cyclin D1 and cyclin E1. The exception to this was the synovial cell lines for which there was no inhibition of p-RB despite inhibition of p-RSK, p-AKT and p-S6. When combined with palbociclib in both the CDK4/6 sensitive (LS141 and DDLS) and CDK4/6 resistant liposarcoma cells (RES.LS141 and RES.DDLS), we observed enhanced inhibition of proliferation with a synergistic effect, as defined by the Chou-Talalay method. These results would suggest a role for PDK-1 targeting with SNS-510 in sarcoma and consideration of combination strategies, especially with CDK4/6 inhibitors in both CDK4/6 sensitive and resistant dedifferentiated liposarcoma. Citation Format: Elgilda Musi, Pietro Taverna, Gary K. Schwartz. PDK-1 targeting with SNS-510 in sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1067.

Abstract 475: Improving liposarcoma diagnosis through cross-flow microfiltration

Abstract Introduction: Dedifferentiated liposarcoma (DDLPS) is frequently diagnosed late and patients typically respondpoorly to treatments bringing to an overall survival rate of only 10% at 10 years. Prognosis isenhanced by early detection of recurrent lesions, but current diagnostic methods rely on invasivetissue biopsies combined with whole or part-body imaging. These diagnostic and prognosticchallenges prompted us to explore the use of circulating biomolecules as potential biomarkersaccessible through bodily fluids. Extracellular Vesicles (EVs) are membrane vesicles containingproteins and nucleic acids that circulate in the blood, so they are an emerging class of easilyaccessible blood biomarkers. We recently identified extracellular vesicle EV-MDM2 as liquidbiopsy biomarker for disease identification in DDLPS. Unfortunately, due to these challenges forisolation and capture of EVs, despite the potential diagnostic and prognostic utility, the practicalrelevance of using EVs for routine analysis remains limited. Methods: A microfluidic device was fabricated via standard soft lithography in PDMS(polydimethylsiloxane). A 5 x 5 mm2 nanocapillary array membrane (nominal capillary diameterof 0.2 µm) was sandwiched between two microchannels for a vertically aligned configuration withan injection and separation channel (each 500 µm wide and 150 µm high) perpendicular to eachother. The separation channel surface was functionalized with the anti-CD63 antibody forcapturing EVs. Results: We present here a facile approach to fabricate and operate a micro-nanofluidic device that usescross-flow filtration to isolate and capture liposarcoma derived EVs. Our methodology integratesthe unit operations of size based separation with immunoaffinity-based capture of extracellularvesicles in the same device. The eluted media collected showed ~84% extracellular vesiclerecovery from the liposarcoma cell conditioned media and ~38% extracellular vesicle recoveryfrom dedifferentiated liposarcoma patient serum when compared to extracellular vesicle isolationand subsequent quantification by ultracentrifugation. The results reported here also show a five-fold increase in amount of critical liposarcoma relevant extracellular vesicle cargo obtained in30 minutes presenting a significant advance over existing state-of-art. Conclusion: In conclusion we developed a cross-flow filtration micro-nanofluidic device for isolation, selectivecapture and release of liposarcoma EVs. Citation Format: Lucia Casadei, Patricia Sarchet, Adarsh Choudhury, Shaurya Prakash, Raphael Pollock. Improving liposarcoma diagnosis through cross-flow microfiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 475.

Crosstalk between Macrophages and Myxoid Liposarcoma Cells Increases Spreading and Invasiveness of Tumor Cells.

Simple SummaryMyxoid liposarcoma (MLPS) is the second most common subtype of liposarcoma, occurs predominantly in the extremities, and tends either to recur locally and metastasize to unusual soft tissue locations. To date, the mechanisms of invasion and metastasis of MLPS remain unclear, and yet, there is a high need to identify new prognostic biomarkers to enable the development of novel targeted therapeutic strategies. This study firstly aimed to assess the role of immune cellular components that infiltrate MLPS tissues. Our data show that high grade, heavily vascularized MLPS tissues exhibit T lymphocyte-poor and M2-like macrophage-rich phenotypes, while low grade MLPS tissues are mainly infiltrated by T lymphocytes. In line with these findings, evidence is shown that a crosstalk occurring between MLPS cells and macrophages exists as MLPS cells drive an M2-like phenotype in monocytes which in turn, increase the invasive capability of MLPS cells.Myxoid liposarcoma (MLPS) is the second most common subtype of liposarcoma and has tendency to metastasize to soft tissues. To date, the mechanisms of invasion and metastasis of MLPS remain unclear, and new therapeutic strategies that improve patients’ outcomes are expected. In this study, we analyzed by immunohistochemistry the immune cellular components and microvessel density in tumor tissues from patients affected by MLPS. In order to evaluate the effects of primary human MLPS cells on macrophage polarization and, in turn, the ability of macrophages to influence invasiveness of MLPS cells, non-contact and 3D organotypic co-cultures were set up. High grade MLPS tissues were found heavily vascularized, exhibited a CD3, CD4, and CD8 positive T lymphocyte-poor phenotype and were massively infiltrated by CD163 positive M2-like macrophages. Conversely, low grade MLPS tissues were infiltrated by a discrete amount of CD3, CD4, and CD8 positive T lymphocytes and a scarce amount of CD163 positive macrophages. Kaplan–Meier analysis revealed a shorter Progression Free Survival in MLPS patients whose tumor tissues were highly vascularized and heavily infiltrated by CD163 positive macrophages, indicating a clear-cut link between M2-like macrophage abundance and poor prognosis in patients. Moreover, we documented that, in co-culture, soluble factors produced by primary human MLPS cells induce macrophage polarization toward an M2-like phenotype which, in turn, increases MLPS cell capability to spread into extracellular matrix and to cross endothelial monolayers. The identification of M2-like polarization factors secreted by MLPS cells may allow to develop novel targeted therapies counteracting MLPS progression.

Promoter G-quadruplexes and transcription factors cooperate to shape the cell type-specific transcriptome

Cell identity is maintained by activation of cell-specific gene programs, regulated by epigenetic marks, transcription factors and chromatin organization. DNA G-quadruplex (G4)-folded regions in cells were reported to be associated with either increased or decreased transcriptional activity. By G4-ChIP-seq/RNA-seq analysis on liposarcoma cells we confirmed that G4s in promoters are invariably associated with high transcription levels in open chromatin. Comparing G4 presence, location and transcript levels in liposarcoma cells to available data on keratinocytes, we showed that the same promoter sequences of the same genes in the two cell lines had different G4-folding state: high transcript levels consistently associated with G4-folding. Transcription factors AP-1 and SP1, whose binding sites were the most significantly represented in G4-folded sequences, coimmunoprecipitated with their G4-folded promoters. Thus, G4s and their associated transcription factors cooperate to determine cell-specific transcriptional programs, making G4s to strongly emerge as new epigenetic regulators of the transcription machinery.

Sarcomas Epidemiology in Iran: Analysis of Data from Iranian National Cancer Registry between 2009 and 2014

Background: Sarcomas are a wide group of tumors with a mesenchymal origin, accounting for 1% of all adult malignancies. Objectives: This study examined the incidence rates and distribution patterns of morphological subtypes and primary sites of soft tissue and bone sarcomas in a 6-years period in the total population of Iran. Methods: The data was retrieved from the Iranian National Cancer Registry (INCR). A total of 14630 patients were identified with either bone or soft tissue sarcoma during 2009 - 2014. The morphological and topographical classifications were based on the ICD-O-3 and World Health Organization (WHO) guidelines. The crude and age-standardized incidence rates (ASIR) were calculated. Results: The combined crude incidence was calculated 3.2/100,000, and for STS and BS was 2.7 and 0.5 per 100,000 individuals, respectively, increasing with the age. Among the patients, 16.47 and 83.53% of cases had BS and STS, respectively with male predilection. The ASIR calculated 2.8 and 2.6 for STS and 0.51 and 0.37 for BS in males and females, respectively. Sarcoma, osteosarcoma, leiomyosarcoma, liposarcoma, and spindle cell sarcoma were the most common morphologies. The connective tissue of the lower limb, long bone of the lower limb, skin, and uterus were the most frequent primary tumor sites. The majority of tumors were of unknown/undifferentiated grade (grade 9), then grade 3, 1, 2, 4, and score 0 had the highest frequencies, respectively. The highest ASIR of sarcoma was detected in Khuzestan, Kohgiloyeh and Boyer-Ahmad, Isfahan, Tehran, Fars, and Khorasan Razavi provinces. Analysis of the incidence trends showed a slight increase over the study period. Conclusions: These findings can help better diagnose and management of sarcoma patients throughout Iran.