Comparison of three-dimensional cell culture techniques of dedifferentiated liposarcoma and their integration with future research.

Background: Dedifferentiated liposarcoma is a formidable sarcoma subtype due to its high local recurrence rate and resistance to medical treatment. While 2D cell cultures are still commonly used, 3D cell culture systems have emerged as a promising alternative, particularly scaffold-based techniques that enable the creation of 3D models with more accurate cell-stroma interactions. Objective: To investigate how 3D structures with or without the scaffold existence would affect liposarcoma cell lines growth morphologically and biologically. Methods: Lipo246 and Lipo863 cell lines were cultured in 3D using four different methods; Matrigel® ECM scaffold method, Collagen ECM scaffold method, ULA plate method and Hanging drop method, in addition to conventional 2D cell culture methods. All samples were processed for histopathological analysis (HE, IHC and DNAscope™), Western blot, and qPCR; moreover, 3D collagen-based models were treated with different doses of SAR405838, a well-known inhibitor of MDM2, and cell viability was assessed in comparison to 2D model drug response. Results: Regarding morphology, cell lines behaved differently comparing the scaffold-based and scaffold-free methods. Lipo863 formed spheroids in Matrigel® but not in collagen, while Lipo246 did not form spheroids in either collagen or Matrigel®. On the other hand, both cell lines formed spheroids using scaffold-free methods. All samples retained liposarcoma characteristic, such as high level of MDM2 protein expression and MDM2 DNA amplification after being cultivated in 3D. 3D collagen samples showed higher cell viability after SAR40538 treatment than 2D models, while cells sensitive to the drug died by apoptosis or necrosis. Conclusion: Our results prompt us to extend our investigation by applying our 3D models to further oncological relevant applications, which may help address unresolved questions about dedifferentiated liposarcoma biology.

Radiation therapy for retroperitoneal sarcoma: practice patterns in North America.

BACKGROUND: The addition of radiation therapy (RT) to surgery in retroperitoneal sarcoma (RPS) remains controversial. We examined practice patterns in the use of RT for patients with RPS over time in a large, national cohort. METHODS: Patients in the National Cancer Database (2004-2017) who underwent resection of RPS were included. Trends over time for proportions were calculated using contingency tables with Cochran-Armitage Trend test. RESULTS: Of 7,485 patients who underwent resection, 1,821 (24.3%) received RT (adjuvant: 59.9%, neoadjuvant: 40.1%). The use of RT decreased annually by < 1% (p = 0.0178). There was an average annual increase of neoadjuvant RT by 13% compared to an average annual decrease of adjuvant RT by 6% (p < 0.0001). Treatment at high-volume centers (OR 14.795, p < 0.0001) and tumor > 10 cm (OR 2.009, p = 0.001) were associated with neoadjuvant RT. In contrast liposarcomas (OR 0.574, p = 0.001) were associated with adjuvant RT. There was no statistically significant difference in overall survival between patients treated with surgery alone versus surgery and RT (p = 0.07). CONCLUSION: In the United States, the use of RT for RPS has decreased over time, with a shift towards neoadjuvant RT. However, a large percentage of patients are still receiving adjuvant RT and this mostly occurs at low-volume hospitals.

Disparities in Survival and NCCN Guideline-Concordant Care in Patients With Extremity Soft Tissue Sarcoma.

BACKGROUND: Based on the NCCN Guidelines for Soft Tissue Sarcoma (STS), treatment of extremity STS (ESTS) includes radiation therapy (RT) and surgical resection for tumors that are high-grade and >5 cm. ​​The aim of this study was to describe the association between neighborhood socioeconomic status (nSES), concordance with NCCN Guidelines recommendations, and outcomes in patients with ESTS. METHODS: Patients with ESTS diagnosed from 2006 through 2018 were identified in SEER registries. The analytic cohort was restricted to patients with high-grade tumors >5 cm without nodal or distant metastases who received limb-sparing surgery. Patient demographics and tumor characteristics associated with receipt of RT were analyzed using adjusted regression analyses. Kaplan-Meier curves and adjusted accelerated failure time models were used to examine disparities in cancer-specific survival. RESULTS: Of 2,249 patients, 29.0% (n=648) received neoadjuvant RT, 49.7% (n=1,111) received adjuvant or intraoperative RT, and 21.3% (n=476) did not receive RT. In adjusted analyses, lower nSES was associated with lower likelihood of receiving RT (odds ratio, 0.70 [95% CI, 0.57-0.87]; P<.001). Low nSES was associated with worse cancer-specific survival (hazard ratio, 1.19 [95% CI, 1.01-1.40]; P=.04). Race and ethnicity were not significant predictors of receipt of RT or cancer-specific survival in the fully adjusted models. CONCLUSIONS: Patients from lower nSES areas were less likely to receive NCCN Guideline-recommended RT for their ESTS and had worse cancer-specific survival. Efforts to better define and resolve disparities in the treatment and survival of patients with ESTS are warranted.

Comparing Multivisceral Resection with Tumor-only Resection of Liposarcoma Using the Win Ratio.

BACKGROUND: Multivisceral resection of retroperitoneal liposarcoma (LPS) is associated with increased morbidity and may not confer a survival benefit compared with tumor-only (TO) resection. We compared both approaches using a novel statistical method called the "win ratio" (WR). METHODS: Patients who underwent resection of LPS from 2004 to 2015 were identified from the National Cancer Database. Multivisceral resection was defined as removal of the primary site in addition to other organs. The WR was calculated based on a hierarchy of postoperative outcomes: 30-day and 90-day mortality, long-term survival, and severe complication. RESULTS: Among 958 patients (multivisceral 634, TO 324) who underwent resection, the median age was 63 years (interquartile range [IQR] 54-71) with a median follow-up of 51 months (IQR 30-86). There was no difference in the WR among patients who underwent TO versus multivisceral resection in the matched cohort (WR 0.82, 95% confidence interval [CI] 0.61-1.10). In patients aged 72-90 years, those who underwent multivisceral resection had 36% lower odds of winning compared with patients undergoing TO resection (WR 0.64, 95% CI 0.40-0.98). A subgroup analysis of patients classified as not having adjacent tumor involvement at the time of surgery revealed that those patients who underwent multivisceral resection had 33% lower odds of winning compared to TO resection (WR 0.67, 95% CI 0.45-0.99). CONCLUSIONS: Based on win-ratio assessments of a hierarchical composite endpoint, multivisceral resection in patients without adjacent tumor involvement may not confer improved outcomes. This method supports the rationale for less invasive resection of LPS in select patients, especially older patients.

Preoperative radiation therapy increases adherence in patients with high-risk extremity soft tissue sarcoma.

BACKGROUND: Surgery and radiation therapy remain the standard of care for patients with high-grade extremity soft tissue sarcoma that are >5 cm. Radiation therapy is time and labor-intensive for patients, and social determinants of health may affect adherence. The aim of this study was to define demographic, clinical, and treatment factors associated with the completion of radiation therapy and determine if preoperative radiation therapy improved adherence compared to postoperative radiation therapy. METHODS: The cohort included patients in the National Cancer Database with high-grade extremity soft tissue sarcoma >5 cm without nodal or distant metastases who received limb-sparing surgery and radiation therapy with microscopically negative R0 margins. Multivariable logistic regression analyses identified factors associated with radiation therapy sequencing and adherence (defined as completion of 50 Gy preoperative radiation therapy or at least 60 Gy postoperative radiation therapy). A multivariable Cox Proportional Hazards model assessed overall survival. RESULTS: Among 2,145 patients, 47.1% received preoperative radiation therapy (n = 1,010), and 52.9% (n = 1135) received postoperative radiation therapy. A greater proportion of patients treated with preoperative (77.2%) versus postoperative radiation therapy (64.9%, P < .0001) received the recommended dose. More patients with private insurance (49.8% vs 35.3% Medicaid vs 44.9% Medicare, P = .011) and patients treated at an academic medical center (52.6% vs 47.4%, P < .001) received preoperative radiation therapy. Patients who received preoperative radiation therapy had lower odds of receiving insufficient doses of radiation therapy (odds ratio 0.34 [95% CI 0.27-0.47]). Neither radiation therapy adherence nor sequencing were independent predictors of overall survival. CONCLUSIONS: Patients who received preoperative radiation therapy were more likely to complete therapy and receive an optimal dose than patients treated with postoperative radiation therapy. Preoperative radiation therapy improves adherence and should be widely considered in patients with high-grade extremity soft tissue sarcoma, particularly in patients at risk for not completing therapy.

Erratum: ATR inhibition sensitizes liposarcoma to doxorubicin by increasing DNA damage.

[This corrects the article on p. 1577 in vol. 12, PMID: 35530299.].

Notch signaling regulates a metabolic switch through inhibiting PGC-1α and mitochondrial biogenesis in dedifferentiated liposarcoma.

Human dedifferentiated liposarcoma (DDLPS) is a rare but lethal cancer with no driver mutations being identified, hampering the development of targeted therapies. We and others recently reported that constitutive activation of Notch signaling through overexpression of the Notch1 intracellular domain (NICDOE) in murine adipocytes leads to tumors resembling human DDLPS. However, the mechanisms underlying the oncogenic functions of Notch activation in DDLPS remains unclear. Here, we show that Notch signaling is activated in a subset of human DDLPS and correlates with poor prognosis and expression of MDM2, a defining marker of DDLPS. Metabolic analyses reveal that murine NICDOE DDLPS cells exhibit markedly reduced mitochondrial respiration and increased glycolysis, mimicking the Warburg effect. This metabolic switch is associated with diminished expression of peroxisome proliferator-activated receptor gamma coactivator 1α (Ppargc1a, encoding PGC-1α protein), a master regulator of mitochondrial biogenesis. Genetic ablation of the NICDOE cassette rescues the expression of PGC-1α and mitochondrial respiration. Similarly, overexpression of PGC-1α is sufficient to rescue mitochondria biogenesis, inhibit the growth and promote adipogenic differentiation of DDLPS cells. Together, these data demonstrate that Notch activation inhibits PGC-1α to suppress mitochondrial biogenesis and drive a metabolic switch in DDLPS.

Adjuvant Palbociclib May be Associated with Delayed Recurrence in Completely Resected Retroperitoneal Liposarcoma: Results of a Single-Institution Retrospective Cohort Study.

BACKGROUND: Retroperitoneal liposarcomas are locally aggressive and frequently recur following complete surgical resection. Palbociclib, a cyclin-dependent kinase (CDK) 4/CDK6 inhibitor, is effective in the treatment of metastatic or unresectable liposarcoma. OBJECTIVE: The purpose of this study was to describe our initial experience using adjuvant palbociclib to delay recurrence. METHODS: Patients with resected RPS were identified from a prospectively maintained institutional database. In 2017, we began offering adjuvant palbociclib to patients following complete gross resection. Treatment interval, defined as the time between surgical resection and re-resection or change in systemic therapy, was compared between patients selected for adjuvant palbociclib or observation. RESULTS: Between 2017 and 2020, 12 patients underwent a total of 14 operations (14 patient cases) and were selected for adjuvant palbociclib for recurrence prevention. These patients were compared with 14 patients who, since 2010, underwent a total of 20 operations (20 patient cases) and were selected for observation. Histology was primarily dedifferentiated liposarcoma for both groups (observation: 70% [14/20]; adjuvant palbociclib: 64% [9/14]). All patients underwent complete gross resection. Neither age, number of previous surgeries, histologic grade, or Eastern Cooperative Oncology Group (ECOG) performance status differed between groups (p > 0.05 for all). Patients selected for adjuvant palbociclib experienced a longer treatment interval than those selected for observation, although it did not reach statistical significance (20.5 months vs. 13.1 months, p = 0.08, log rank). CONCLUSION: Adjuvant palbociclib may be associated with a prolonged interval between liposarcoma resection and the need for re-resection or other systemic therapy. Palbociclib may be effective in delaying liposarcoma recurrence, and its use for this indication warrants prospective study.

Phosphorylation of IWS1 by AKT maintains liposarcoma tumor heterogeneity through preservation of cancer stem cell phenotypes and mesenchymal-epithelial plasticity.

Chemotherapy remains the mainstay of treatment for patients with advanced liposarcoma (LPS), but response rates are only 25% and the overall survival at 5 years is dismal at 20-34%. Translation of other therapies have not been successful and there has been no significant improvement in prognosis for nearly 20 years. The aberrant activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway has been implicated in the aggressive clinical behavior LPS and in resistance to chemotherapy, but the precise mechanism remains elusive and efforts to target AKT clinically have failed. Here we show that the AKT-mediated phosphorylation of the transcription elongation factor IWS1, promotes the maintenance of cancer stem cells in both cell and xenograft models of LPS. In addition, phosphorylation of IWS1 by AKT contributes to a "metastable" cell phenotype, characterized by mesenchymal/epithelial plasticity. The expression of phosphorylated IWS1 also promotes anchorage-dependent and independent growth, cell migration, invasion, and tumor metastasis. In patients with LPS, IWS1 expression is associated with reduced overall survival, increased frequency of recurrence, and shorter time to relapse after resection. These findings indicate that IWS1-mediated transcription elongation is an important regulator of human LPS pathobiology in an AKT-dependent manner and implicate IWS1 as an important molecular target to treat LPS.

Small Non-Coding RNAs in Soft-Tissue Sarcomas: State of the Art and Future Directions.

Soft-tissue sarcomas (STS) are a rare and heterogeneous group of tumors that arise from connective tissue and can occur anywhere in the body. Among the plethora of over 50 different STS types, liposarcoma (LPS) is one of the most common. The subtypes of STS are characterized by distinct differences in tumor biology that drive responses to pharmacologic therapy and disparate oncologic outcomes. Small non-coding RNAs (sncRNA) are a heterogeneous class of regulatory RNAs involved in the regulation of gene expression by targeting mRNAs. Among the several types of sncRNAs, miRNAs and tRNA-derived ncRNAs are the most studied in the context of tumor biology, and we are learning more about the role of these molecules as important regulators of STS tumorigenesis and differentiation. However, challenges remain in translating these findings and no biomarkers or therapeutic approaches targeting sncRNAs have been developed for clinical use. In this review, we summarize the current landscape of sncRNAs in the context of STS with an emphasis on LPS, including the role of sncRNAs in the tumorigenesis and differentiation of these rare malignancies and their potential as novel biomarkers and therapeutic targets. Finally, we provide an appraisal of published studies and outline future directions to study sncRNAs in STS, including tRNA-derived ncRNAs.

Emerging Trends in Immunotherapy for Adult Sarcomas.

Soft-tissue sarcomas (STS) are a rare and heterogeneous group of malignant tumors that arise from the oncogenic transformation of mesenchymal tissue. There are over 100 distinct STS histological and molecular subtypes with unique clinical, therapeutic, and prognostic features with variable responses to therapy regimens. Given the quality-of-life concerns and limited efficacy with current regimens, including cytotoxic chemotherapy, there is a need for novel therapies and regimens to treat advanced STS. Although immune checkpoint inhibitors have demonstrated significant improvements in survival outcomes in other cancer types, there remains ambiguous data on the impact of immunotherapy in sarcoma. Biomarkers like PD-1/PD-L1 are not always predictive of outcomes. Therefore, researching emerging novel therapies, such as CAR-T and adoptive cell therapies, is critical to understanding STS biology, STS tumor immune microenvironment immunomodulatory strategies that improve immune response, and survival outcomes. We discuss the underlying biology of the STS tumor immune microenvironment, immunomodulatory strategies that augment pre-existing immune responses, and novel approaches to develop sarcoma-specific antigen-based therapies.

Three dimensional models of dedifferentiated liposarcoma cell lines: scaffold-based and scaffold-free approaches.

Sarcomas are rare malignancies, the number of reports is limited, and this rarity makes further research difficult even though liposarcoma is one of major sarcomas. 2D cell culture remains an important role in establishing basic tumor biology research, but its various shortcomings and limitations are still of concern, and it is now well-accepted that the behavior of 3D-cultured cells is more reflective of in vivo cellular responses compared to 2D models. This study aimed to establish 3D cell culture of liposarcomas using two different methods: scaffold-based (Matrigel extracellular matrix [ECM] scaffold method) and scaffold-free (Ultra-low attachment [ULA] plate). Lipo246, Lipo224 and Lipo863 cell lines were cultured, and distinctive differences in structures were observed in Matrigel 3D model: Lipo224 and Lipo863 formed spheroids, whereas Lipo246 grew radially without forming spheres. In ULA plate approaches, all cell lines formed spheroids, but Lipo224 and Lipo863 spheroids showed bigger size and looser aggregation than Lipo246. Formalin fixed, paraffin embedded (FFPE) blocks were obtained from all 3D models, confirming the spheroid structures. The expression of MDM2, Ki-67 positivity and MDM2 amplification were confirmed by IHC and DNAscope™, respectively. Protein and DNA were extracted from all samples and MDM2 upregulation was confirmed by western blot and qPCR analysis. After treatment with MDM2 inhibitor SAR405838, DDLPS spheroids demonstrated different sensitivity patterns from 2D models. Taken together, we believed that 3D models would have a possibility to provide us a new predictability of efficacy and toxicity, and considered as one important process in in vitro pre-clinical phase prior to moving forward to clinical trials.

Enhancer Coamplification and Hijacking Promote Oncogene Expression in Liposarcoma.

SIGNIFICANCE: Comprehensive profiling of the enhancer landscape and 3D genome structure in liposarcoma identifies extensive enhancer-oncogene coamplification and enhancer hijacking events, deepening the understanding of how oncogenes are regulated in cancer.

Prognostic factors in patients receiving surgery and radiation therapy for retroperitoneal sarcoma: A machine-learning analysis.

BACKGROUND: The addition of radiation therapy to surgery for retroperitoneal sarcoma remains controversial. Improved patient selection may help identify optimal candidates for multimodality treatment. The aim of this analysis was to define prognostic factors among patients who receive radiation therapy and surgery to aid in patient selection for multimodal therapy. METHODS: Patients who received radiation therapy and underwent curative-intent resection for retroperitoneal sarcoma between 2004 and 2016 were identified from a national cohort in the United States (National Cancer Database). A machine-based classification and regression tree model was used to generate similar groups of patients relative to overall survival based on preoperative factors. RESULTS: A total of 1,443 patients received radiation therapy in addition to surgery. Median age was 61 years old and 55.0% were female. Most patients (66%) received care at an academic or integrated network cancer program. With a median follow-up of 84 months, receipt of radiation therapy was not associated with improved overall survival (P = .81). Classification and regression tree analysis revealed a significant association between overall survival and American Joint Committee on Cancer stage group, age, tumor histology, and Charlson comorbidity score. Application of these parameters via machine learning stratified patients into 5 cohorts with distinct survival outcomes. In the most favorable cohort (Cohort 1: American Joint Committee on Cancer stage group ≤II, age ≤61, histology including fibrosarcoma, well differentiated liposarcoma, myxoid liposarcoma, and leiomyosarcoma), the 5-year overall survival was 81.7% and median overall survival was not reached; in the least favorable cohort (Cohort 6: American Joint Committee on Cancer stage group >II, age >68) where the 5-year survival was 41.3% and median overall survival was 45.2 months (P < .001 versus Cohort 1). CONCLUSION: In the absence of a defined survival benefit, patients with advanced American Joint Committee on Cancer stage group, older age, and medical comorbidities have relatively unfavorable overall survival after combined modality therapy and therefore stand the least to gain from the addition of radiation therapy to surgery. In contrast, younger patients with good performance status and retroperitoneal sarcoma histologies with a higher propensity for local recurrence may have the greatest opportunity to benefit from radiation therapy.

Preclinical efficacy of the Wnt/ß-catenin pathway inhibitor BC2059 for the treatment of desmoid tumors.

Mutation in the CTNNB1 gene, leading to a deregulation of the WTN/ß-catenin pathway, is a common feature of desmoid tumors (DTs). Many ß-catenin inhibitors have recently been tested in clinical studies; however, BC2059 (also referred as Tegavivint), a selective inhibitor of nuclear ß-catenin that works through binding TBL-1, is the only one being evaluated in a clinical study, specifically for treatment of desmoid tumor patients. Preclinical studies on BC2059 have shown activity in multiple myeloma, acute myeloid leukemia and osteosarcoma. Our preclinical studies provide data on the efficacy of BC2059 in desmoid cell lines, which could help provide insight regarding antitumor activity of this therapy in desmoid tumor patients. In vitro activity of BC2059 was evaluated using desmoid tumor cell lines. Ex vivo activity of BC2059 was assessed using an explant tissue culture model. Pharmacological inhibition of the nuclear ß-catenin activity using BC2059 markedly inhibited cell viability, migration and invasion of mutated DT cells, but with lower effect on wild-type DTs. The decrease in cell viability of mutated DT cells caused by BC2059 was due to apoptosis. Treatment with BC2059 led to a reduction of ß-catenin-associated TBL1 in all mutated DT cells, resulting in a reduction of nuclear ß-catenin. mRNA and protein levels of AXIN2, a ß-catenin target gene, were also found to be downregulated after BC2059 treatment. Taken together, our results demonstrate that nuclear ß-catenin inhibition using BC2059 may be a novel therapeutic strategy for desmoid tumor treatment, especially in patients with CTNNB1 mutation.

In situ hybridization to detect DNA amplification in extracellular vesicles.

EVs have emerged as an important component in tumour initiation, progression and metastasis. Although notable progresses have been made, the detection of EV cargoes remain significantly challenging for researchers to practically use; faster and more convenient methods are required to validate the EV cargoes, especially as biomarkers. Here we show, the possibility of examining embedded EVs as substrates to be used for detecting DNA amplification through ultrasensitive in situ hybridization (ISH). This methodology allows the visualization of DNA targets in a more direct manner, without time consuming optimization steps or particular expertise. Additionally, formalin-fixed paraffin-embedded (FFPE) blocks of EVs allows long-term preservation of samples, permitting future studies. We report here: (i) the successful isolation of EVs from liposarcoma tissues; (ii) the EV embedding in FFPE blocks (iii) the successful selective, specific ultrasensitive ISH examination of EVs derived from tissues, cell line, and sera; (iv) and the detection of MDM2 DNA amplification in EVs from liposarcoma tissues, cell lines and sera. Ultrasensitive ISH on EVs would enable cargo study while the application of ISH to serum EVs, could represent a possible novel methodology for diagnostic confirmation. Modification of probes may enable researchers to detect targets and specific DNA alterations directly in tumour EVs, thereby facilitating detection, diagnosis, and improved understanding of tumour biology relevant to many cancer types.

The Role of AKT in Soft Tissue Sarcoma: Review and Insights.

Soft tissue sarcomas (STS) are a biologically diverse group of mesenchymal tumors that predominantly exhibit a poor prognosis. Surgical resection is considered the mainstay of treatment and provides the only chance for long-term survival. However, some patients present with locally advanced, unresectable disease, and for those who are able to undergo resection, tumor recurrence occurs in over half of patients. In addition, the efficacy of conventional systemic therapies remains dismal. The serine/threonine kinase AKT pathway is one of the most frequently aberrantly activated signaling pathways that has been verified in many types of human cancer. Dysregulation of the AKT cascade is known to result in tumorigenesis and aggressive clinical behavior for many tumor types, including STS. EGFRs, with its downstream effectors, PI3K and protein kinase B (AKT)/mTOR, have been investigated for decades as promising targets for the treatment of STS, but significant challenges remain and the prognosis of patients with advanced STS has not improved in over two decades. In this review, we will first describe the AKT pathway and its role in STS tumor biology and then discuss the current challenges in targeting the AKT pathway to treat patients with advanced sarcoma.

Clinical genomic profiling in the management of patients with soft tissue and bone sarcoma.

There are more than 70 distinct sarcomas, and this diversity complicates the development of precision-based therapeutics for these cancers. Prospective comprehensive genomic profiling could overcome this challenge by providing insight into sarcomas' molecular drivers. Through targeted panel sequencing of 7494 sarcomas representing 44 histologies, we identify highly recurrent and type-specific alterations that aid in diagnosis and treatment decisions. Sequencing could lead to refinement or reassignment of 10.5% of diagnoses. Nearly one-third of patients (31.7%) harbor potentially actionable alterations, including a significant proportion (2.6%) with kinase gene rearrangements; 3.9% have a tumor mutational burden ≥10 mut/Mb. We describe low frequencies of microsatellite instability (<0.3%) and a high degree of genome-wide loss of heterozygosity (15%) across sarcomas, which are not readily explained by homologous recombination deficiency (observed in 2.5% of cases). In a clinically annotated subset of 118 patients, we validate actionable genetic events as therapeutic targets. Collectively, our findings reveal the genetic landscape of human sarcomas, which may inform future development of therapeutics and improve clinical outcomes for patients with these rare cancers.