Effects of bone marrow mesenchymal stem cell-conditioned medium on the proliferation and migration of liposarcoma cells.

INTRODUCTION: Liposarcoma constitutes a prevalent subtype of soft tissue sarcoma, represents approximately 20% of all sarcomas. However, conventional chemotherapeutic agents have shown restricted effectiveness in treating liposarcoma patients. Accumulating evidence indicates that mesenchymal stem cells (MSCs) have the characteristic of migration to tumor site, promote or suppress tumors. How human bone marrow mesenchymal stem cells (BMSCs) contribute to liposarcoma phenotype remains poorly understood. This study aims to investigate the effects of human bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) on the proliferation and migration of liposarcoma cell lines 93T449 and SW872, as well as explore potential underlying mechanisms of BMSC-CM action on these cells. MATERIALS AND METHODS: We transfected BMSCs with lentiviral constructs to knock down the transcriptional co-activator Yes-associated protein 1 (YAP1), conditioned medium (CM) obtained from BMSCs and shYAP1-BMSC, respectively. Liposarcoma cell lines 93T449 and SW872 were co-cultured with BMSC-CM or shYAP1-BMSC-CM. Cell proliferation ability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was evaluated using flow cytometric assay. A wound healing assay was used to analyze cell migration. The expression levels of YAP1, Bcl-2, and matrix metalloproteinase-2 (MMP-2) were determined by western blot assay. RESULTS: Co-culturing liposarcoma cell lines 93T449 and SW872 with BMSC-CM promoted tumor cell proliferation, while shYAP1-BMSC-CM significantly inhibited cell viability and migration, induced apoptosis, and downregulated Bcl-2 and MMP-2 expression. CONCLUSIONS: These findings provide new insights into the impact of BMSC-CM on liposarcoma and suggest its possible involvement in liposarcoma cell growth.

Clozapine suppresses NADPH oxidase activation, counteracts cytosolic H2O2, and triggers early onset mitochondrial dysfunction during adipogenesis of human liposarcoma SW872 cells.

Long-term treatment of schizophrenia with clozapine (CLZ), an atypical antipsychotic drug, is associated with an increased incidence of metabolic disorders mediated by poorly understood mechanisms. We herein report that CLZ, while slowing down the morphological changes and lipid accumulation occurring during SW872 cell adipogenesis, also causes an early (day 3) inhibition of the expression/nuclear translocation of CAAT/enhancer-binding protein ß and peroxisome proliferator-activated receptor γ. Under the same conditions, CLZ blunts NADPH oxidase-derived reactive oxygen species (ROS) by a dual mechanism involving enzyme inhibition and ROS scavenging. These effects were accompanied by hampered activation of the nuclear factor (erythroid-derived2)-like 2 (Nrf2)-dependent antioxidant responses compared to controls, and by an aggravated formation of mitochondrial superoxide. CLZ failed to exert ROS scavenging activities in the mitochondrial compartment but appeared to actively scavenge cytosolic H2O2 derived from mitochondrial superoxide. The early formation of mitochondrial ROS promoted by CLZ was also associated with signs of mitochondrial dysfunction. Some of the above findings were recapitulated using mouse embryonic fibroblasts. We conclude that the NADPH oxidase inhibitory and cytosolic ROS scavenging activities of CLZ slow down SW872 cell adipogenesis and suppress their Nrf2 activation, an event apparently connected with increased mitochondrial ROS formation, which is associated with insulin resistance and metabolic syndrome. Thus, the cellular events characterised herein may help to shed light on the more detailed molecular mechanisms explaining some of the adverse metabolic effects of CLZ.

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.

Expression and function of Siglec-15 in RLPS and its correlation with PD-L1: Bioinformatics Analysis and Clinicopathological Evidence.

Purpose: Retroperitoneal liposarcoma (RLPS) is a rare malignancy without effective treatment. Since current treatment for unresectable RLPS is unsatisfactory, immunotherapy and targeted therapy are urgently needed. Siglec-15 is a transmembrane protein highly homologous to PD-L1 and is involved in tumor immune escape. The biological function of Siglec-15 in RLPS, its prognostic relevance and its relationship with PD-L1 need to be further clarified. In this study, we aimed to explore the biological function of Siglec-15 in sarcomas through bioinformatics analysis, and we also evaluated Siglec-15 and PD-L1 expression in RLPS samples. The relationship between the expression of Siglec-15 and PD-L1 and their clinicopathological relevance and prognostic value were also investigated in clinical RLPS patients. Methods: The RNA sequencing data of 259 sarcoma cases and 48 RLPS cases from TCGA were used to analyze the Siglec-15 expression and the differentially expressed genes (DEG) related with Siglec-15 expression. In addition, DEGs were subsequently analyzed through the gene ontology (GO)/ Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) network. Tumor specimens were obtained from 91 RLPS patients of our sarcoma center, and Siglec-15 and PD-L1 expression were evaluated using immunohistochemistry. The correlation between the expression level of these two markers as well as their correlation with clinicopathological factors and prognosis of RLPS patients was also assessed. Results: GEPIA analysis showed that the high expression of Siglec-15 was associated with poor sarcoma OS (P=0.034). A total of 682 differential genes were identified between the high and low expression groups of Siglec-15 in RLPS. Enrichment analysis of the KEGG pathway showed that Siglec-15 was related to the Hippo signaling pathway and the neuroactive ligand-receptor interaction. GO annotation analysis showed that the expression of Siglec-15 may thus be able to affect serine hydrolase activity, alongside signal receptor activator activity. The top 5 genes with the largest number of connection points are APOA1, F2, AHSG, AMBP, SERPINC1. In subsequent studies, we used 91 liposarcoma samples from our center for verification. Siglec-15 was expressed in 84.6% of RLPS cases, whereas PD-L1 was expressed in 17.6% of RLPS cases. A negative correlation was observed between Siglec-15 and PD-L1 expression (P=0.020). In this group of RLPS patients, high Siglec-15 expression was correlated with poorer disease-free survival (DFS) (P=0.021), and it was an independent predictor of DFS (hazard ratio: 2.298; 95% confidence interval: 1.154-4.576; P=0.018). However, we did not find a correlation between PD-L1 expression and overall survival or DFS in RLPS patients. Conclusion: The DEG and signaling pathways identified in the study could provide a preliminary understanding of the underlying molecular mechanisms of Siglec-15 in the development and progression of RLPS. High expression of Siglec-15 was a negative independent predictive factor for DFS of RLPS. The negative relationship between Siglec-15 and PD-L1 expression suggested that the Siglec-15 pathway might be an important supplement to PD-L1 treatment.

Transcriptome analysis identifies TODL as a novel lncRNA associated with proliferation, differentiation, and tumorigenesis in liposarcoma through FOXM1.

Liposarcoma, the most common soft tissue sarcoma, is a group of fat cell mesenchymal tumors with different histological subtypes. The dysregulation of long non-coding RNAs (lncRNAs) has been observed in human cancers including a few studies in sarcoma. However, the global transcriptome analysis and potential role of lncRNAs remain unexplored in liposarcoma. The present investigation uncovers the transcriptomic profile of liposarcoma by RNA sequencing to gain insight into the global transcriptional changes in liposarcoma. Our RNA sequencing analysis has identified that many oncogenic lncRNAs are differentially expressed in different subtypes of liposarcoma including MALAT1, PVT1, SNHG15, LINC00152, and MIR210HG. Importantly, we identified a highly overexpressed, unannotated, and novel lncRNA in dedifferentiated liposarcomas. We have named it TODL, transcript overexpressed in dedifferentiated liposarcoma. TODL lncRNA displayed significantly higher expression in dedifferentiated liposarcoma cell lines and patient samples. Interestingly, functional studies revealed that TODL lncRNA has an oncogenic function in liposarcoma cells by regulating proliferation, cell cycle, apoptosis, differentiation, and tumorigenesis in the murine model. Silencing of TODL lncRNA highlighted the enrichment of several key oncogenic signaling pathways including cell cycle, transcriptional misregulation, FOXM1 network, p53 signaling, PLK1 signaling, FoxO, and signaling Aurora signaling pathways. RNA pull-down assay revealed the binding of TODL lncRNA with FOXM1, an oncogenic transcription factor, and the key regulator of the cell cycle. Silencing of TODL lncRNA also induces adipogenesis in dedifferentiated liposarcomas. Altogether, our finding indicates that TODL could be utilized as a novel, specific diagnostic biomarker, and a pharmacological target for therapeutic development in controlling aggressive and metastatic dedifferentiated liposarcomas.

Growth-Suppressive and Apoptosis-Inducing Effects of Tetrandrine in SW872 Human Malignant Liposarcoma Cells via Activation of Caspase-9, Down-Regulation of XIAP and STAT-3, and ER Stress.

Liposarcoma is a rare and heterogeneous soft tissue malignant tumor and has a significant impact on mortality with a poor prognosis. To date, there is no effective treatment for liposarcoma, whereas surgical resection is only the gold treatment with numerous adverse effects. Here we investigated whether tetrandrine inhibits the growth of SW872 human malignant liposarcoma cells. Of note, tetrandrine at 10 µM vastly inhibited growth and induced apoptosis, as evidenced by increased nuclear DNA fragmentation and sub-G1 population of SW872 cells. Mechanistically, treatment with tetrandrine led to activation of caspase-9/3 in SW872 cells, and z-VAD-fmk, a pan-caspase inhibitor, attenuated the tetrandrine-induced apoptosis and growth suppression in SW872 cells. In addition, tetrandrine treatment resulted in down-regulation of XIAP andSTAT-3 in SW872 cells, and importantly knockdown of STAT-3 caused a significant reduction of the cell survival. Tetrandrine also had abilities to up-regulate not only the expression of GRP78 and ATF-4 but also the phosphorylation of eIF-2α in SW872 cells. In summary, these results demonstrated that tetrandrine has strong growth-suppressive and apoptosis-inducing effects on SW872 cells, which are mediated through control of the intrinsic caspase pathway, down-regulation of XIAP and STAT-3, and triggering ER stress.

Discrimination of lipoma from atypical lipomatous tumor/well-differentiated liposarcoma using magnetic resonance imaging radiomics combined with machine learning.

PURPOSE: To evaluate the diagnostic capability of radiomics in distinguishing lipoma and Atypic Lipomatous Tumors/Well-Differentiated Liposarcomas (ALT/WDL) with Magnetic Resonance Imaging (MRI). MATERIALS AND METHODS: Patients with a histopathologic diagnosis of lipoma (n = 45) and ALT/WDL (n = 20), who had undergone pre-surgery or pre-biopsy MRI, were enrolled. The MDM2 amplification was accepted as gold-standard test. The T1-weighted turbo spin echo images were used for radiomics analysis. Utility of a predefined standardized imaging protocol and a single type of 1.5 T scanner were sought as inclusion criteria. Radiomics parameters that show a certain level of reproducibility were included in the study and supplied to Support Vector Machine (SVM) as a machine learning method. RESULTS: No significant difference was found in terms of gender, location and age between the lipoma and ALT/WDL groups. Sixty-five parameters were accepted as reproducible. Fifty-seven parameters were able to distinguish the two groups significantly (AUC range 0.564-0.902). Diagnostic performance of the SVM was one of the highest among literature findings: sensitivity = 96.8% (95% CI 94.03-98.39%), specificity = 93.72% (95% CI 86.36-97.73%) and AUC = 0.987 (95% CI 0.972-0.999). CONCLUSION: Although radiomics has been proven to be useful in previous literature regarding discrimination of lipomas and ALT/WDLs, we found that its accuracy could further be improved with utility of standardized hardware, imaging protocols and incorporation of machine learning methods.

Negative MDM2/CDK4 immunoreactivity does not fully exclude MDM2/CDK4 amplification in a subset of atypical lipomatous tumor/ well differentiated liposarcoma.

Our main goal was to investigate the potential utility of MDM2/CDK4 immunohistochemistry to act as surrogate for FISH to identify a subset of lipoma-like ALT/WDL that might, otherwise, be underdiagnosed on initial screening. Lack of cytologic atypia in lipomatous tumors with negative expression for MDM2/CDK4 IHC does not fully exclude the possibility of underlying MDM2/CDK4 amplification. Present study identified that isolated CDK4/p16 positive expression, in the absence of MDM2 expression, may have potential utility during the initial screening of these tumors but the proportion of conventional lipomas, which may also exhibit low levels of CDK4/p16 expression remains uncertain.

Lessons learnt from MDM2 fluorescence in-situ hybridisation analysis of 439 mature lipomatous lesions with an emphasis on atypical lipomatous tumour/well-differentiated liposarcoma lacking cytological atypia.

AIMS: Amplification of the murine double minute-2 (MDM2) gene, which is usually detected with fluorescence in-situ hybridisation (FISH), is the key driving event for atypical lipomatous tumours (ALTs)/well-differentiated liposarcomas (WDLs). We sought to determine the concordance between the histopathological findings and MDM2 FISH in the diagnosis of ALT/WDL, and to identify the histological features of MDM2-amplified tumours lacking classic atypia. METHODS AND RESULTS: We performed a retrospective analysis of all mature lipomatous lesions subjected to MDM2 FISH analysis at our institution. MDM2 FISH analysis was performed on 439 mature lipomatous lesions: 364 (82.9%) were negative and 75 (17%) were positive. In 17 of 75 (22.6%) ALTs/WDLs, cytological atypia was not identified on initial histological assessment, thus favouring lipoma. On review, these cases shared common histological features, consisting of a very low number of relatively small stromal cells within the tumour lobules, with mildly coarse chromatin and oval nuclei, admixed with unremarkable adipocytes in a tumour background devoid of fibroconnective septa, areas of fibrosis, or blood vessels. These cells matched the cells in which FISH showed MDM2 amplification. In contrast, 13 cases (3.5%) regarded as suspicious for ALT/WDL on the basis of histology lacked MDM2 amplification and were reclassified following the FISH findings. CONCLUSIONS: We conclude that a subset of lipoma-like ALTs/WDLs are not associated with any of the features typically described in ALT/WDL. Our study also showed that tumours >100 mm are more likely to be ALT/WDL; however, a history of recurrence or concerning clinical/radiological features was not significantly associated with classification as ALT/WDL.

Targeting abundant survivin expression in liposarcoma: subtype dependent therapy responses to YM155 treatment.

PURPOSE: Liposarcoma (LPS) represent the largest group of malignant soft tissue tumours comprising a heterogeneous group of subtypes in which the degrees of chemoresistance and radiosensitivity strongly vary. Consequently, it is of utmost interest to establish novel therapeutic regimens based on molecular targets. METHODS: Immunohistochemical staining of survivin was performed in tissue microarrays comprising 49 primary LPS specimens. LPS cell lines were treated with survivin antagonist YM155 and doxorubicin or etoposide alone as well as in combination. Changes in cell viability were investigated and the synergistic effect of a combined therapy analysed. RESULTS: Immunohistochemistry revealed an abundant expression of survivin in LPS that significantly concurred with less-differentiated tumour subtypes and grading. In vitro, we demonstrated the impact of the survivin inhibitor YM155 on dedifferentiated LPS (DDLPS) and, even more imposing, pleomorphic LPS (PLS) tumour cell viability with a strong induction of apoptosis. A combined treatment of doxorubicin or etoposide with YM155 augmented the cytotoxic effects on DDLPS and PLS cells. CONCLUSION: These findings support the significant role of survivin in the oncogenesis and progression of LPS subtypes providing a rationale to target survivin in eligible in-vivo models and to pioneer clinical applications of survivin-specific substances unfolding their therapeutic potential in LPS patients prospectively.

Expression of FRS2 in atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma: an immunohistochemical analysis of 182 cases with genetic data.

BACKGROUND: The fibroblast growth factor receptor substrate 2 (FRS2) gene is located close to MDM2 and CDK4 within the 12q13-15 chromosomal region. FRS2 gene was recently found to be consistently amplified in atypical lipomatous tumor (ALT)/well-differentiated liposarcoma (WDL) and dedifferentiated liposarcoma (DDL), suggesting the detection of FRS2 amplification could be a diagnostic tool for ALT/WDL/DDLs. However, the expression of FRS2 protein and diagnostic value of FRS2 immunohistochemistry (IHC) has not been evaluated in a large cohort of ALT/WDL/DDLs. METHODS: A SNOMED search of hospital surgical pathology files from January 2007 to July 2020 identified 182 ALT/WDL/DDLs with available materials. FRS2 fluorescence in situ hybridization (FISH) and IHC were performed on 182 ALT/WDL/DDLs and 64 control samples. The expression of FRS2 was also compared with that of classic immunomarkers (MDM2 and CDK4) of this tumor entity. RESULTS: This study included 91 ALT/WDLs and 91 DDLs. The FISH results showed 172 of 182 (94.5%) cases were FRS2-amplified, and 10 cases were FRS2-nonamplified. Immunostaining results showed 171 (94.0%) ALT/WDL/DDLs were positive for FRS2 and 11 cases (6.0%) were FRS2-immunonegative. In 172 FRS2-amplified cases, 166 (96.5%) were FRS2-immunopositive, and 6 (3.5%) were negative. Among 10 FRS2-nonamplified ALT/WDL/DDL cases, 5 cases were FRS2-immunonegative, and 5 tumors displayed 1+ staining for this marker. In 64 control cases, none of them exhibited FRS2 amplification. Forty-seven (73.5%) control cases were negative for FRS2 immunostaining, while 17 cases (26.5%) were FRS2-immunopositive. Fifteen of these false positive samples (15/17, 88.2%) showed 1+ positivity and only 2 cases (2/17, 11.8%) displayed 2+ positivity. In ALT/WDL/DDLs, the sensitivity of FRS2 immunostaining was slightly lower than MDM2 (FRS2 vs. MDM2: 94.0% vs 100.0%) and CDK4 (FRS2 vs. CDK4: 94.0% vs 97.0%). However, the specificity of FRS2 (73.5%) was slightly higher than that of MDM2 (67.8%) and CDK4 (64.4%). CONCLUSION: This study indicated that FRS2 IHC had relatively good consistency with FRS2 FISH, suggesting that FRS2 immunostaining could be utilized as an additional screening tool for the diagnosis of ALT/WDL/DDL. It must be emphasized that MDM2/CDK4/FRS2 especially MDM2 FISH remains the gold standard and the most recommended method to diagnose this entity.

Clear Cell Proliferations of the Skin: A Histopathologic Review.

ABSTRACT: Cutaneous clear cell proliferations encompass a heterogenous group of several primary cutaneous neoplasms and metastatic tumors with different histogenesis. Many of these clear cell proliferations may seem strikingly similar under the microscope resulting in challenging diagnosis. In many of these clear cell lesions, the reason for the clear or pale appearance of proliferating cells is unknown, whereas in other ones, this clear cell appearance is due to intracytoplasmic accumulation of glycogen, mucin, or lipid. Artifacts of tissue processing and degenerative phenomenon may also be responsible for the clear cell appearance of proliferating cells. Awareness of the histopathologic findings as well as histochemical and immunohistochemical techniques are crucial to the accurate diagnosis. This review details the histopathologic features of clear cell cutaneous proliferations, classifying them according their type of differentiation and paying special attention to the histopathologic differential diagnosis among them.

Myxoid pleomorphic liposarcoma-a clinicopathologic, immunohistochemical, molecular genetic and epigenetic study of 12 cases, suggesting a possible relationship with conventional pleomorphic liposarcoma.

Myxoid pleomorphic liposarcoma is a recently defined subtype of liposarcoma, which preferentially involves the mediastinum of young patients and shows mixed histological features of conventional myxoid liposarcoma and pleomorphic liposarcoma. While myxoid pleomorphic liposarcoma is known to lack the EWSR1/FUS-DDIT3 fusions characteristic of the former, additional genetic data are limited. To further understand this tumor type, we extensively examined a series of myxoid pleomorphic liposarcomas by fluorescence in situ hybridization (FISH), shallow whole genome sequencing (sWGS) and genome-wide DNA methylation profiling. The 12 tumors occurred in 6 females and 6 males, ranging from 17 to 58 years of age (mean 33 years, median 35 years), and were located in the mediastinum (n = 5), back, neck, cheek and leg, including thigh. Histologically, all cases consisted of relatively, bland, abundantly myxoid areas with a prominent capillary vasculature, admixed with much more cellular and less myxoid foci containing markedly pleomorphic spindled cells, numerous pleomorphic lipoblasts and elevated mitotic activity. Using sWGS, myxoid pleomorphic liposarcomas were found to have complex chromosomal alterations, including recurrent large chromosomal gains involving chromosomes 1, 6-8, 18-21 and losses involving chromosomes 13, 16 and 17. Losses in chromosome 13, in particular loss in 13q14 (including RB1, RCTB2, DLEU1, and ITM2B genes), were observed in 4 out of 8 cases analyzed. Additional FISH analyses confirmed the presence of a monoallelic RB1 deletion in 8/12 cases. Moreover, nuclear Rb expression was deficient in all studied cases. None showed DDIT3 gene rearrangement or MDM2 gene amplification. Using genome-wide DNA methylation profiling, myxoid pleomorphic liposarcomas and conventional pleomorphic liposarcomas formed a common methylation cluster, which segregated from conventional myxoid liposarcomas. While the morphologic, genetic and epigenetic characteristics of myxoid pleomorphic liposarcoma suggest a link with conventional pleomorphic liposarcoma, its distinctive clinical features support continued separate classification for the time being.

Update on genomic and molecular landscapes of well-differentiated liposarcoma and dedifferentiated liposarcoma.

Well-differentiated liposarcoma (WDLPS) is the most frequent subtype of liposarcoma and may transform into dedifferentiated liposarcoma (DDLPS) which is a more aggressive subtype. Retroperitoneal lesions of WDLPS/DDLPS tend to recur repeatedly due to incomplete resections, and adjuvant chemotherapy and radiotherapy have little effect on patient survival. Consequently, identifying therapeutic targets and developing targeted drugs is critical for improving the outcome of WDLPS/DDLPS patients. In this review, we summarised the mutational landscape of WDLPS/DDLPS from recent studies focusing on potential oncogenic drivers and the development of molecular targeted drugs for DDLPS. Due to the limited number of studies on the molecular networks driving WDLPS to DDLPS development, we looked at other dedifferentiation-related tumours to identify potential parallel mechanisms that could be involved in the dedifferentiation process generating DDLPS.

Clinical Application of Chromosome Microarray Analysis in the Diagnosis of Lipomatous Tumors.

Well-differentiated liposarcoma/atypical lipomatous tumor (WDLS/ALT) and dedifferentiated liposarcoma (DDLS) have characteristic supernumerary ring and giant marker chromosomes involving the chromosomal region 12q13-15 which contains MDM2 (12q15), CDK4 (12q14.1), HMGA2 (12q14.3), YEATS4 (12q15), CPM (12q15), and FRS2 (12q15). Detecting MDM2 amplification by fluorescence in situ hybridization (FISH) is considered to be the gold standard for the diagnosis of WDLS/ALT and DDLS. In this study, formalin fixed paraffin embedded clinical specimens (16 liposarcomas and 19 benign lipomatous tumors) were used to detect MDM2 amplification and other chromosomal alterations in WDLS/ALT and DDLS by single nucleotide polymorphism-based chromosome microarray (CMA). All 16 liposarcomas showed MDM2 amplification with a MDM2/cep12 ratio from 2.4 to 8.4 by CMA. Ten (62.5%) of these cases had CDK4/cep12 ratio ≥2.0. All the cases without CDK4 amplification were from the thigh. The MDM2/cep12 ratio of all the benign lipomatous tumors (19/19) was within the normal limits. Twenty-one of the 35 benign lipomatous tumors and liposarcomas were also tested for MDM2 amplification by FISH. All the FISH results were consistent with the CMA results (100%). Along with MDM2 amplification, all 16 liposarcomas (100%) also showed amplification of YEATS4, CPM and FRS2. Only 11 of 16 (69%) cases showed HMGA2 amplification. In conclusion, this study demonstrated that CMA on routine formalin fixed paraffin embedded tissue is a sensitive and specific clinical test for detection of MDM2 gene amplification. Moreover, CMA allows simultaneous detection of genomic changes of interest including CDK4 and others, which provides enriched information for diagnosing lipomatous tumors.

Selinexor versus doxorubicin in dedifferentiated liposarcoma PDXs: evidence of greater activity and apoptotic response dependent on p53 nuclear accumulation and survivin down-regulation.

BACKGROUND: Dedifferentiated liposarcoma (DDLPS), a tumor that lacks effective treatment strategies and is associated with poor outcomes, expresses amplified MDM2 in the presence of wild-type p53. MDM2 ubiquitination of p53 facilitates its XPO1-mediated nuclear export, thus limiting p53 tumor suppressor functions. Consequently, nuclear export is a rational target in DDLPS. We directly compared the antitumor activity of the first-in class XPO1 inhibitor selinexor and doxorubicin, the standard front-line therapy in sarcomas, in DDLPS patient-derived xenografts (PDXs) and primary cell lines. METHODS: Drug activity was assessed in three PDXs (and two corresponding cell lines) established from the dedifferentiated component of primary untreated retroperitoneal DDLPS with myogenic (N = 2) and rhabdomyoblastic (N = 1) differentiation from patients who underwent surgery. These models were marked by amplification of MDM2, CDK4 and HMGA2 genes. RESULTS: Selinexor was moderately active in the three PDXs but achieved greater tumor response compared to doxorubicin (maximum tumor volume inhibition: 46-80 % vs. 37-60 %). The PDX harboring rhabdomyoblastic dedifferentiation showed the highest sensitivity to both agents. PDX response to selinexor and doxorubicin was not associated with the extent of MDM2 and CDK4 gene amplification. Interestingly, the most chemosensitive PDX model showed the lowest extent of HMGA2 amplification. Selinexor was also more efficient than doxorubicinin in inducing an apoptotic response in PDXs and cell lines. Consistently, an increased nuclear accumulation of p53 was seen in all selinexor-treated models. In addition, a time-dependent decrease of survivin expression, with an almost complete abrogation of the cytoplasmic anti-apoptotic pool of this protein, was observed as a consequence of the decreased acetylation/activation of STAT3 and the increased ubiquitination of nuclear survivin. CONCLUSIONS: Selinexor showed a moderate antitumor activity in three DDLPS PDXs, which was, however, consistently higher than doxorubicin across all different models regardless the extent of MDM2 amplification and the histological differentiation. The depletion of survivin protein seems to significantly contribute to the induction of apoptosis through which selinexor exerts its antitumor activity.

Frequent amplification of HDAC genes and efficacy of HDAC inhibitor chidamide and PD-1 blockade combination in soft tissue sarcoma.

BACKGROUND: The advent of immune checkpoint therapy has been a tremendous advance in cancer treatment. However, the responses are still insufficient in patients with soft tissue sarcoma (STS). We aimed to identify rational combinations to increase the response to immune checkpoint therapy and improve survival. METHODS: Whole-exome sequencing (WES) was performed in 11 patients with liposarcoma. Somatic copy number alterations (SCNAs) were analyzed at the gene level to identify obvious amplification patterns in drug-target genes. The expression and prognostic value of class I histone deacetylases (HDACs) was evaluated in 49 patients with sarcoma in our center and confirmed in 263 sarcoma samples from The Tumor Cancer Genome Atlas (TCGA) database. Q-PCR, flow cytometry and RNA-seq were performed to determine the correlations between class I HDACs, chidamide and PD-L1 in vitro and in vivo. The efficacy of combining chidamide with PD-1 blockade was explored in an immunocompetent murine model and a small cohort of patients with advanced sarcoma. Western blot, ChIP assay and dual luciferase assessment were applied in the mechanistic study. RESULTS: The HDAC gene family was frequently amplified in STS. SCNAs in the HDAC gene family were extensively amplified in 8 of 11 (73%) patients with liposarcoma, based on a drug-target gene set, and we verified amplification in 76.65% (197/257) of cases by analyzing TCGA sarcoma cohort. Class I HDAC expression is associated with a poor prognosis for patients with STS, and its inhibition is responsible for promoting apoptosis and upregulating of programmed cell death ligand 1 (PD-L1). The HDAC class I inhibitor chidamide significantly increases PD-L1 expression, increased the infiltration of CD8+ T cells and reduced the number of MDSCs in the tumor microenvironment. The combination of chidamide with an anti-PD-1 antibody significantly promotes tumor regression and improves survival in a murine model. Moreover, chidamide combined with the anti-PD-1 antibody toripalimab is effective in patients with advanced and metastatic sarcoma, and the side effects are tolerable. Mechanistically, chidamide increases histone acetylation at the PD-L1 gene through the activation of the transcriptional factor STAT1. CONCLUSIONS: The combination of chidamide and anti-programmed cell death 1 (PD-1) therapy represents a potentially important strategy for STS.

CD10 (neprilysin) expression: a potential adjunct in the distinction of hibernoma from morphologic mimics.

Although the morphologic diagnosis of hibernoma is usually straightforward, some hibernomas have atypical morphologic features, mimicking atypical lipomatous tumors/well-differentiated liposarcomas (ALT/WDLs). In addition, the multivacuolated brown fat cells may be mistaken for lipoblasts by pathologists, especially those without significant soft tissue tumor exposure. Thus, we continue to receive in consultation cases of hibernoma sent for MDM2 fluorescence in situ hybridization testing to exclude ALT/WDL. Testing hibernomas for MDM2 amplification, however, adds cost and delays the final diagnosis. Recently, we have noted expression of neprilysin (CD10, CALLA), a zinc-dependent metalloproteinase involved in the inactivation of various peptide hormones, in brown fat cells, and wished to explore the potential utility of this widely available, inexpensive ancillary test in the differential diagnosis of hibernoma. Formalin-fixed, paraffin-embedded tissue sections from well-characterized cases of hibernoma (n = 48), brown fat (n = 21), ALTs/WDLs (n = 17), pleomorphic liposarcomas (PLPSs) (n = 6), lipomas (n = 5), and fat necrosis (n = 5) were immunostained for CD10, using a commercially available antibody and routine laboratory protocols. CD10 expression was evaluated in both adipocytes and in surrounding stromal cells. The hibernomas occurred in 28 men and 20 women, ranging from 11 to 76 years of age and involved the extremities (n = 25), pelvis (n = 7), abdomen/pelvis/retroperitoneum (n = 7), head and neck region (n = 6), back (n = 2), and chest (n = 1). All showed diffuse, strong CD10 expression in multivacuolated brown fat cells and in the majority of adjacent univacuolated fat cells. Brown adipose tissue from various anatomic structures showed an identical pattern of immunoreactivity. In contrast, CD10 expression was present in the adipocytes of only 3 of 17 (18%) ALTs/WDLs and was absent in lipomas and fat necrosis. Lipoblasts expressed CD10 in 3 PLPSs. Expression of CD10 by surrounding fibroblastic stromal cells was more widespread, present in 13 hibernomas, 10 ALTs/WDLs, 1 instance of fat necrosis, 6 PLPSs, and 4 examples of brown fat. We conclude that immunohistochemistry for CD10 may represent a useful, rapid and inexpensive ancillary test in the differential diagnosis of hibernoma from potential morphologic mimics, especially when morphologic features favor hibernoma. CD10 expression in adipocytes, however, should be rigorously distinguished from fibroblastic stromal cell CD10 expression, a nonspecific finding.

Anti-Survival and Pro-Apoptotic Effects of 6-Shogaol on SW872 Human Liposarcoma Cells via Control of the Intrinsic Caspase Pathway, STAT-3, AMPK, and ER Stress.

Notably, 6-Shogaol, a bioactive natural substance, has anticancer effects on many types of tumors. Up to date, the anticancer effect and mode of action of 6-Shogaol on liposarcoma are not known. In this study, we investigated whether 6-Shogaol inhibits the growth of SW872 and 93T449 cells, two different human liposarcoma cell lines. Of note, 6-Shogaol inhibited the growth of SW872 and 93T449 cells without affecting that of normal 3T3-L1 preadipocytes. Specifically, 6-Shogaol further induced the apoptosis of SW872 cells, as evidenced by nuclear DNA fragmentation, increased sub G1 population, activation of the intrinsic caspase pathway, and PARP cleavage. However, pretreatment with either z-VAD-fmk, a pan-caspase inhibitor, or N-acetylcysteine, an antioxidant, attenuated the 6-Shogaol's growth-suppressive and apoptosis-inducing effects on SW872 cells. Moreover, 6-Shogaol activated AMPK while inhibited STAT-3 in SW872 cells, and siRNA-based genetic silencing of AMPK or STAT-3 considerably blocked the growth-suppressive and apoptotic response of 6-Shogaol to SW872 cells. Moreover, 6-Shogaol also upregulated the expression and phosphorylation of GRP-78, eIF-2α, ATF4, and CHOP, known ER stress markers, in SW872 cells, illustrating the induction of ER stress. These findings collectively demonstrate that 6-Shogaol has strong antigrowth and proapoptotic effects on SW872 cells through regulation of the intrinsic caspase pathway, oxidative stress, STAT-3, AMPK, and ER stress.