RESUMO
The detection of disialoganglioside GD2 on tumor biopsies, especially in paraffin-embedded tissues, has been challenging due to the glycolipid structure of GD2 and its membrane anchorage. Here, we present an immunofluorescence protocol for the reliable assessment of GD2 on formalin-fixed paraffin-embedded (FFPE) tissues. We describe steps for antigen retrieval with Tris-EDTA buffer and staining with unconjugated anti-GD2 antibody (clone 14.G2a) and horse radish peroxidase (HRP)-conjugated secondary antibody. We then detail procedures for signal amplification using the tyramide signal amplification technique. For complete details on the use and execution of this protocol, please refer to Fischer-Riepe et al.1.
RESUMO
Undifferentiated small round cell sarcomas (USRS) of bone and soft tissue are a group of tumors with heterogenic genomic alterations sharing similar morphology. In the present study, we performed a comparative large-scale proteomic analysis of USRS (n = 42) with diverse genomic translocations including classic Ewing sarcomas with EWSR1::FLI1 fusions (n = 24) or EWSR1::ERG fusions (n = 4), sarcomas with an EWSR1 rearrangement (n = 2), CIC::DUX4 fusion (n = 8), as well as tumors classified as USRS with no genetic data available (n = 4). Proteins extracted from formalin-fixed, paraffin-embedded pretherapeutic biopsies were analyzed qualitatively and quantitatively using shotgun mass spectrometry (MS). More than 8000 protein groups could be quantified using data-independent acquisition. Unsupervised hierarchical cluster analysis based on proteomic data allowed stratification of the 42 cases into distinct groups reflecting the different molecular genotypes. Protein signatures that significantly correlated with the respective genomic translocations were identified and used to generate a heatmap of all 42 sarcomas with assignment of cases with unknown molecular genetic data to either the EWSR1- or CIC-rearranged groups. MS-based prediction of sarcoma subtypes was molecularly confirmed in 2 cases where next-generation sequencing was technically feasible. MS also detected proteins routinely used in the immunohistochemical approach for the differential diagnosis of USRS. BCL11B highly expressed in Ewing sarcomas, and BACH2 as well as ETS-1 highly expressed in CIC::DUX4-associated sarcomas, were among proteins identified by the present proteomic study, and were chosen for immunohistochemical confirmation of MS data in our study cohort. Differential expressions of these 3 markers in the 2 genetic groups were further validated in an independent cohort of n = 34 USRS. Finally, our proteomic results point toward diverging signaling pathways in the different USRS subgroups.
Assuntos
Biomarcadores Tumorais , Proteômica , Proteína EWS de Ligação a RNA , Sarcoma de Células Pequenas , Translocação Genética , Humanos , Proteína EWS de Ligação a RNA/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/análise , Sarcoma de Células Pequenas/genética , Sarcoma de Células Pequenas/patologia , Sarcoma de Células Pequenas/diagnóstico , Feminino , Masculino , Adulto , Adolescente , Adulto Jovem , Neoplasias de Tecidos Moles/genética , Neoplasias de Tecidos Moles/patologia , Neoplasias de Tecidos Moles/diagnóstico , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/diagnóstico , Criança , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genéticaRESUMO
Carbohydrate markers of immature cells during prenatal human development can be aberrantly expressed in cancers and deserve evaluation as immune targets. A candidate target in Ewing sarcoma is the globo-series ganglioside stage-specific embryonic antigen-4 (SSEA-4). We detected SSEA-4 expression on the cell surface of all of 14 EwS cell lines and in 21 of 31 (68%) primary EwS tumor biopsies. Among paired subpopulations of tumor cells with low versus high SSEA-4 expression, SSEA-4high expression was significantly and consistently associated with functional characteristics of tumor aggressiveness, including higher cell proliferation, colony formation, chemoresistance and propensity to migrate. SSEA-4low versus SSEA-4high expression was not related to expression levels of the EWSR1-FLI1 fusion transcript or markers of epithelial/mesenchymal plasticity. SSEA-4low cells selected from bulk populations regained higher SSEA-4 expression in vitro and during in vivo tumor growth in a murine xenograft model. T cells engineered to express SSEA-4-specific chimeric antigen receptors (CARs) specifically interacted with SSEA-4 positive EwS cells and exerted effective antigen-specific tumor cell lysis in vitro. In conclusion, with its stable expression and functional significance in EwS, SSEA-4 is an attractive therapeutic immune target in this cancer that deserves further evaluation for clinical translation.
Assuntos
Sarcoma de Ewing , Antígenos Embrionários Estágio-Específicos , Animais , Feminino , Humanos , Camundongos , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/patologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Gangliosídeos , Glicoesfingolipídeos , Sarcoma de Ewing/patologia , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/genética , Antígenos Embrionários Estágio-Específicos/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Cytokine-engineering of chimeric antigen receptor-redirected T cells (CAR T cells) is a promising principle to overcome the limited activity of canonical CAR T cells against solid cancers. EXPERIMENTAL DESIGN: We developed an investigational medicinal product, GD2IL18CART, consisting of CAR T cells directed against ganglioside GD2 with CAR-inducible IL18 to enhance their activation response and cytolytic effector functions in the tumor microenvironment. To allow stratification of patients according to tumor GD2 expression, we established and validated immunofluorescence detection of GD2 on paraffin-embedded tumor tissues. RESULTS: Lentiviral all-in-one vector engineering of human T cells with the GD2-specific CAR with and without inducible IL18 resulted in cell products with comparable proportions of CAR-expressing central memory T cells. Production of IL18 strictly depends on GD2 antigen engagement. GD2IL18CART respond to interaction with GD2-positive tumor cells with higher IFNγ and TNFα cytokine release and more effective target cytolysis compared with CAR T cells without inducible IL18. GD2IL18CART further have superior in vivo antitumor activity, with eradication of GD2-positive tumor xenografts. Finally, we established GMP-compliant manufacturing of GD2IL18CART and found it to be feasible and efficient at clinical scale. CONCLUSIONS: These results pave the way for clinical investigation of GD2IL18CART in pediatric and adult patients with neuroblastoma and other GD2-positive cancers (EU CT 2022- 501725-21-00). See related commentary by Locatelli and Quintarelli, p. 3361.
Assuntos
Gangliosídeos , Imunoterapia Adotiva , Interleucina-18 , Neoplasias , Receptores de Antígenos Quiméricos , Linfócitos T , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Gangliosídeos/imunologia , Interleucina-18/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Camundongos , Imunoterapia Adotiva/métodos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Neoplasias/imunologia , Neoplasias/terapia , Neoplasias/patologia , Linhagem Celular Tumoral , Microambiente Tumoral/imunologia , FemininoRESUMO
For more than 20 years gastrointestinal stromal tumors (GIST) have been a paradigm for a targeted treatment with tyrosine kinase inhibitors. A fundamental prerequisite for a neoadjuvant or adjuvant treatment of localized GIST or an additive treatment of metastatic GIST is the molecular typing of tumors, ideally at the initial diagnosis. In addition, the possibility of a hereditary or syndromic predisposition must be considered because this results in consequences for the treatment and a different follow-up strategy.
Assuntos
Tumores do Estroma Gastrointestinal , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/terapia , Humanos , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/patologia , Neoplasias Gastrointestinais/terapia , Neoplasias Gastrointestinais/diagnóstico , Neoplasias Gastrointestinais/tratamento farmacológico , Antineoplásicos/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/genéticaRESUMO
Background: Ewing sarcoma (EwS) is a rare and highly malignant bone tumor primarily affecting children, adolescents, and young adults. The pelvis, trunk, and lower extremities are the most common sites, while EwS of the sacrum as a primary site is very rare, and only few studies focusing on this location are published. Due to the anatomical condition, local treatment is challenging in sacral malignancies. We analyzed factors that might influence the outcome of patients suffering from sacral EwS. Methods: We retrospectively analyzed data of the GPOH EURO-E.W.I.N.G 99 trial and the EWING 2008 trial, with a cohort of 124 patients with localized or metastatic sacral EwS. The study endpoints were overall survival (OS) and event-free survival (EFS). OS and EFS were calculated using the Kaplan-Meier method, and univariate comparisons were estimated using the log-rank test. Hazard ratios (HRs) with respective 95% confidence intervals (CIs) were estimated in a multivariable Cox regression model. Results: The presence of metastases (3y-EFS: 0.33 vs. 0.68; P < 0.001; HR = 3.4, 95% CI 1.7 to 6.6; 3y-OS: 0.48 vs. 0.85; P < 0.001; HR = 4.23, 95% CI 1.8 to 9.7), large tumor volume (≥200 ml) (3y-EFS: 0.36 vs. 0.69; P=0.02; HR = 2.1, 95% CI 1.1 to 4.0; 3y-OS: 0.42 vs. 0.73; P=0.04; HR = 2.1, 95% CI 1.03 to 4.5), and age ≥18 years (3y-EFS: 0.41 vs. 0.60; P=0.02; HR = 2.6, 95% CI 1.3 to 5.2; 3y-OS: 0.294 vs. 0.59; P=0.01; HR = 2.92, 95% CI 1.29 to 6.6) were revealed as adverse prognostic factors. Conclusion: Young age seems to positively influence patients` survival, especially in patients with primary metastatic disease. In this context, our results support other studies, stating that older age has a negative impact on survival. Tumor volume, metastases, and the type of local therapy modality have an impact on the outcome of sacral EwS. Level of evidence: Level 2. This trial is registered with NCT00020566 and NCT00987636.
RESUMO
ABSTRACT: Epigenetic modulation of the cell-intrinsic immune response holds promise as a therapeutic approach for leukemia. However, current strategies designed for transcriptional activation of endogenous transposons and subsequent interferon type-I (IFN-I) response, show limited clinical efficacy. Histone lysine methylation is an epigenetic signature in IFN-I response associated with suppression of IFN-I and IFN-stimulated genes, suggesting histone demethylation as key mechanism of reactivation. In this study, we unveil the histone demethylase PHF8 as a direct initiator and regulator of cell-intrinsic immune response in acute myeloid leukemia (AML). Site-specific phosphorylation of PHF8 orchestrates epigenetic changes that upregulate cytosolic RNA sensors, particularly the TRIM25-RIG-I-IFIT5 axis, thereby triggering the cellular IFN-I response-differentiation-apoptosis network. This signaling cascade largely counteracts differentiation block and growth of human AML cells across various disease subtypes in vitro and in vivo. Through proteome analysis of over 200 primary AML bone marrow samples, we identify a distinct PHF8/IFN-I signature in half of the patient population, without significant associations with known clinically or genetically defined AML subgroups. This profile was absent in healthy CD34+ hematopoietic progenitor cells, suggesting therapeutic applicability in a large fraction of patients with AML. Pharmacological support of PHF8 phosphorylation significantly impairs the growth in samples from patients with primary AML. These findings provide novel opportunities for harnessing the cell-intrinsic immune response in the development of immunotherapeutic strategies against AML.
Assuntos
Epigênese Genética , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/metabolismo , Animais , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Camundongos , Interferon Tipo I/metabolismo , Autorrenovação Celular , Regulação Leucêmica da Expressão GênicaRESUMO
Linking clinical multi-omics with mechanistic studies may improve the understanding of rare cancers. We leverage two precision oncology programs to investigate rhabdomyosarcoma with FUS/EWSR1-TFCP2 fusions, an orphan malignancy without effective therapies. All tumors exhibit outlier ALK expression, partly accompanied by intragenic deletions and aberrant splicing resulting in ALK variants that are oncogenic and sensitive to ALK inhibitors. Additionally, recurrent CKDN2A/MTAP co-deletions provide a rationale for PRMT5-targeted therapies. Functional studies show that FUS-TFCP2 blocks myogenic differentiation, induces transcription of ALK and truncated TERT, and inhibits DNA repair. Unlike other fusion-driven sarcomas, TFCP2-rearranged tumors exhibit genomic instability and signs of defective homologous recombination. DNA methylation profiling demonstrates a close relationship with undifferentiated sarcomas. In two patients, sarcoma was preceded by benign lesions carrying FUS-TFCP2, indicating stepwise sarcomagenesis. This study illustrates the potential of linking precision oncology with preclinical research to gain insight into the classification, pathogenesis, and therapeutic vulnerabilities of rare cancers.
Assuntos
Sarcoma , Neoplasias de Tecidos Moles , Humanos , Multiômica , Medicina de Precisão , Fatores de Transcrição/genética , Sarcoma/genética , Sarcoma/terapia , Sarcoma/diagnóstico , Proteína EWS de Ligação a RNA/genética , Neoplasias de Tecidos Moles/genética , Neoplasias de Tecidos Moles/terapia , Receptores Proteína Tirosina Quinases , Biomarcadores Tumorais/genética , Proteínas de Fusão Oncogênica/genética , Proteína-Arginina N-Metiltransferases , Proteínas de Ligação a DNA/genéticaRESUMO
PURPOSE: The phase III, open-label, prospective, multicenter, randomized Ewing 2008R1 trial (EudraCT2008-003658-13) was conducted in 12 countries to evaluate the effect of zoledronic acid (ZOL) maintenance therapy compared with no add-on regarding event-free survival (EFS, primary endpoint) and overall survival (OS) in standard-risk Ewing sarcoma (EWS). PATIENTS AND METHODS: Eligible patients had localized EWS with either good histologic response to induction chemotherapy and/or small tumors (<200 mL). Patients received six cycles of VIDE induction and eight cycles of VAI (male) or eight cycles of VAC (female) consolidation. ZOL treatment started parallel to the sixth consolidation cycle. Randomization was stratified by tumor site (pelvis/other). The two-sided adaptive inverse-normal four-stage design (planned sample size 448 patients, significance level 5%, power 80%) was changed after the first interim analysis using the Müller-Schäfer method. RESULTS: Between April 2010 and November 2018, 284 patients were randomized (142 ZOL/142 no add-on). With a median follow-up of 3.9 years, EFS was not significantly different between ZOL and no add-on group in the adaptive design (HR, 0.74; 95% CI, 0.43-1.28, P = 0.27, intention-to-treat). Three-year EFS rates were 84.0% (95% CI, 77.7%-90.8%) for ZOL vs. 81.7% (95% CI, 75.2%-88.8%) for no add-on. Results were similar in the per-protocol collective. OS was not different between groups. The 3-year OS was 92.8% (95% CI, 88.4%-97.5%) for ZOL and 94.6% (95% CI, 90.9%-98.6%) for no add-on. Noticeable more renal, neurologic, and gastrointestinal toxicities were observed for ZOL (P < 0.05). Severe renal toxicities occurred more often in the ZOL arm (P = 0.003). CONCLUSIONS: In patients with standard-risk localized EWS, there is no additional benefit from maintenance treatment with ZOL.
Assuntos
Neoplasias Ósseas , Sarcoma de Ewing , Humanos , Masculino , Feminino , Sarcoma de Ewing/patologia , Ácido Zoledrônico/uso terapêutico , Estudos Prospectivos , Intervalo Livre de Progressão , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias Ósseas/patologiaRESUMO
Osseous lesions are rare; however, their incidence is increased in childhood and adolescence. The spectrum of osseous processes in this age group is limited, with benign lesions being much more prevalent than malignant tumors. For the differential diagnosis, it is essential to have in-depth knowledge of the more frequent bone diseases in children and adolescents. The current review presents these diseases based on the morphologic approach of the WHO classification, including giant cell-rich and cystic lesions, chondrogenic and bone-forming lesions [7]. Small round cell sarcomas which are now summarized in a separate chapter of the WHO classification have been described previously [12, 20].
Assuntos
Neoplasias Ósseas , Humanos , Adolescente , Criança , Neoplasias Ósseas/diagnóstico , Células Gigantes/patologiaRESUMO
Giant cell tumor of bone (GCTB) is an osteolytic tumor driven by an H3F3A-mutated mononuclear cell with the accumulation of osteoclastic giant cells. We analyzed tissue from 13 patients with recurrence and 25 patients with denosumab therapy, including two cases of malignant transformation. We found a decrease in the total number of cells (p = 0.03), but not in the individual cell populations when comparing primary and recurrence. The patients treated with denosumab showed induction of osteoid formation increasing during therapy. The total number of cells was reduced (p < 0.0001) and the number of H3F3A-mutated tumor cells decreased (p = 0.0001), while the H3F3A wild-type population remained stable. The KI-67 proliferation rate dropped from 10% to 1% and Runx2- and SATB2-positive cells were reduced. The two cases of malignant transformation revealed a loss of the H3F3A-mutated cells, while the KI-67 rate increased. Changes in RUNX2 and SATB2 expression were higher in one sarcoma, while in the other RUNX2 was decreased and SATB2-positive cells were completely lost. We conclude that denosumab has a strong impact on the morphology of GCTB. KI-67, RUNX2 and SATB2 expression differed depending on the benign or malignant course of the tumor under denosumab therapy.
RESUMO
BACKGROUND: Local treatment is a crucial element in the standard of care for Ewing sarcoma (EWS). While systemic treatment is improved in randomised clinical trials, local treatment modalities are discussed controversially. We analysed the association between local therapy and event-free survival (EFS), overall survival (OS), and local recurrence (LR) in prospectively collected data of patients with localised EWS. PATIENTS AND METHODS: We analysed data from the international Ewing 2008 study registered between 2009 and 2019 in 117 centres. After induction chemotherapy, patients received surgery, radiotherapy, or a combination thereof. We performed Cox regression, conducted propensity score-weighted sensitivity analysis, and performed subgroup analyses. Hazard ratios (HRs) and 95% confidence intervals are reported. RESULTS: We included 863 patients with localised EWS (surgery alone: 331, combination therapy: 358, definitive radiotherapy: 174). In patients treated with combination therapy compared to surgery alone, EFS HR was 0.84 (0.57-1.24; p = 0.38), OS HR was 0.84 (0.57-1.23; p = 0.41), and LR HR was 0.58 (0.26-1.31; p = 0.19). Hazards of any event were increased in patients treated with definitive radiotherapy compared to surgery only, HR 1.53 (1.02-2.31; p = 0.04). Patients with poor responses to chemotherapy benefitted from combination therapy over definitive surgery with an EFS HR 0.49 (0.27-0.89; p = 0.02). Patients with pelvic tumours benefitted from combination therapy over surgery only regarding LR, HR 0.12 (0.02-0.72; p = 0.02). CONCLUSION: Patients with poor responses to chemotherapy benefitted from radiotherapy added to surgery. In the whole group, radiotherapy alone as opposed to surgery alone increased the hazards of any event.
Assuntos
Radioterapia (Especialidade) , Sarcoma de Ewing , Humanos , Sarcoma de Ewing/terapia , Intervalo Livre de Progressão , Terapia Combinada , Quimioterapia de InduçãoRESUMO
Purpose: Radiation therapy (RT) is an integral part of Ewing sarcoma (EwS) therapy. The Ewing 2008 protocol recommended RT doses ranging from 45 to 54 Gy. However, some patients received other doses of RT. We analyzed the effect of different RT doses on event-free survival (EFS) and overall survival (OS) in patients with EwS. Methods and Materials: The Ewing 2008 database included 528 RT-admitted patients with nonmetastatic EwS. Recommended multimodal therapy consisted of multiagent chemotherapy and local treatment consisting of surgery (S&RT group) and/or RT (RT group). EFS and OS were analyzed with uni- and multivariable Cox regression models including known prognostic factors such as age, sex, tumor volume, surgical margins, and histologic response. Results: S&RT was performed in 332 patients (62.9%), and 145 patients (27.5%) received definitive RT. Standard dose ≤ 53 Gy (d1) was admitted in 57.8%, high dose of 54 to 58 Gy (d2) in 35.5%, and very high dose ≥ 59 Gy (d3) in 6.6% of patients. In the RT group, RT dose was d1 in 11.7%, d2 in 44.1%, and d3 in 44.1% of patients. Three-year EFS in the S&RT group was 76.6% for d1, 73.7% for d2, and 68.2% for d3 (P = .42) and in the RT group 52.9%, 62.5%, and 70.3% (P = .63), respectively. Multivariable Cox regression revealed age ≥ 15 years (hazard ratio [HR], 2.68; 95% confidence interval [CI], 1.63-4.38) and nonradical margins (HR, 1.76; 95% CI, 1.05-2.93) for the S&RT group (sex, P = .96; histologic response, P = .07; tumor volume, P = .50; dose, P = .10) and large tumor volume (HR, 2.20; 95% CI, 1.21-4.0) for the RT group as independent factors (dose, P = .15; age, P = .08; sex, P = .40). Conclusions: In the combined local therapy modality group, treatment with higher RT dose had an effect on EFS, whereas higher dose of radiation when treated with definitive RT was associated with an increased OS. Indications for selection biases for dosage were found. Upcoming trials will assess the value of different RT doses in a randomized manner to control for potential selection bias.
RESUMO
To enhance the potency of chimeric antigen receptor (CAR) engineered T cells in solid cancers, we designed a novel cell-based combination strategy with an additional therapeutic mode of action. CAR T cells are used as micropharmacies to produce a targeted pro-coagulatory fusion protein, truncated tissue factor (tTF)-NGR, which exerts pro-coagulatory activity and hypoxia upon relocalization to the vascular endothelial cells that invade tumor tissues. Delivery by CAR T cells aimed to induce locoregional tumor vascular infarction for combined immune-mediated and hypoxic tumor cell death. Human T cells that were one-vector gene-modified to express a GD2-specific CAR along with CAR-inducible tTF-NGR exerted potent GD2-specific effector functions while secreting tTF-NGR that activates the extrinsic coagulation pathway in a strictly GD2-dependent manner. In murine models, the CAR T cells infiltrated GD2-positive tumor xenografts, secreted tTF-NGR into the tumor microenvironment and showed a trend towards superior therapeutic activity compared with control cells producing functionally inactive tTF-NGR. In vitro evidence supports a mechanism of hypoxia-mediated enhancement of T cell cytolytic activity. We conclude that combined CAR T cell targeting with an additional mechanism of antitumor action in a one-vector engineering strategy is a promising approach to be further developed for targeted treatment of solid cancers.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Animais , Camundongos , Linfócitos T , Células Endoteliais , Linhagem Celular Tumoral , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Morte Celular , Hipóxia/metabolismo , Imunoterapia Adotiva , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias/terapia , Neoplasias/metabolismoRESUMO
Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide and its most important risk factor is tobacco smoking. While smoking is associated with inferior outcome in NSCLC patients, smoking also correlates with a higher tumor mutational burden. In contrast to adenocarcinomas (ADC) of non-smokers, that frequently harbor targetable gain-of-function mutations, NSCLC smokers largely present with non-targetable loss-of-function mutations of genes associated with DNA-damage repair. The transcription factor Pit-1, Oct1/2, Unc-86 (POU) domain class 2 transcription factor 1 (POU2F1) is a widely expressed bipotential stabilizer of repressed and inducible transcriptional states and frequently deregulated in cancer. Methods: Via immunohistochemistry, we evaluated POU2F1 protein expression on a tissue micro array of 217 operable stage I-III NSCLC patients. Findings were reproduced in a gene expression database of 1144 NSCLC patients, filtered for POU2F1 mRNA expression. After retroviral overexpression of POU2F1 in A549 cells, we evaluated for clonogenic growth and proliferation. Additionally, CRISPR-Cas9 mediated POU2F1 knockdown in A549 cells was likewise analyzed. Results: High protein expression of POU2F1 in 217 NSCLC patients resulted in improved outcome of smokers with ADC [hazard ratio (HR) 0.30 (0.09-0.99), P=0.035]. Moreover, gene expression analysis confirmed favorable outcome of high POU2F1 mRNA expression in smokers with ADC [HR 0.41 (0.24-0.69), P<0.001]. Other than that, retrovirally induced overexpression of POU2F1 in A549 cells significantly reduced both, clonogenic growth as well as proliferation of NSCLC cells, whereas CRISPR-Cas9 mediated knockdown of the protein did not have any impact. Conclusions: Our data suggest that high expression of POU2F1 mediates a less aggressive cancer phenotype in smokers with ADC NSCLC. Pharmacological induction of genes and signaling pathways controlled by POU2F1 may provide novel avenues for future targeted NSCLC therapies in smokers.
RESUMO
Mesenchymal chondrosarcoma (MCS) is a rare and highly aggressive tumour of soft tissue and bone that is defined by an underlying and highly specific fusion transcript involving HEY1 and NCOA2. Histologically, the tumours show a biphasic appearance consisting of an undifferentiated blue and round cell component as well as islands of highly differentiated cartilage. Particularly in core needle biopsies, the chondromatous component can be missed and the non-specific morphology and immunophenotype of the round cell component can cause diagnostic challenges. We applied NKX3.1 immunohistochemistry which was recently reported as a highly specific marker as well as methylome and copy number profiling to a set of 45 well characterised MCS cases to evaluate their potential diagnostic value. Methylome profiling revealed a highly distinct cluster for MCS. Notably, the findings were reproducible also when analysing the round cell and cartilaginous component separately. Furthermore, four outliers were identified by methylome profiling for which the diagnosis had to be revised. NKX3.1 immunohistochemistry showed positivity in 36% of tumours, the majority of which was rather focal and weak. Taken together, NKX3.1 expression showed a low sensitivity but a high specificity in our analysis. Methylome profiling on the other hand represents a sensitive, specific and reliable tool to support the diagnosis of MCS, particularly if only the round cell component is obtained in a biopsy and the diagnosis is not suspected. Furthermore, it can aid in confirming the diagnosis in case RNA sequencing for the HEY1::NCOA2 fusion transcript is not available.
Assuntos
Neoplasias Ósseas , Condrossarcoma Mesenquimal , Humanos , Condrossarcoma Mesenquimal/diagnóstico , Condrossarcoma Mesenquimal/genética , Condrossarcoma Mesenquimal/patologia , Imuno-Histoquímica , Epigenoma , Osso e Ossos/patologia , Diferenciação Celular , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologiaRESUMO
Resection margin adequacy plays a critical role in the local control of sarcomas. Fluorescence-guided surgery has increased complete resection rates and local recurrence-free survival in several oncological disciplines. The purpose of this study was to determine whether sarcomas exhibit sufficient tumor fluorescence (photodynamic diagnosis (PDD)) after administration of 5-aminolevulinic acid (5-ALA) and whether photodynamic therapy (PDT) has an impact on tumor vitality in vivo. Sixteen primary cell cultures were derived from patient samples of 12 different sarcoma subtypes and transplanted onto the chorio-allantoic membrane (CAM) of chick embryos to generate 3-dimensional cell-derived xenografts (CDXs). After treatment with 5-ALA, the CDXs were incubated for another 4 h. Subsequently accumulated protoporphyrin IX (PPIX) was excited by blue light and the intensity of tumor fluorescence was analyzed. A subset of CDXs was exposed to red light and morphological changes of both CAMs and tumors were documented. Twenty-four hours after PDT, the tumors were excised and examined histologically. High rates of cell-derived engraftments on the CAM were achieved in all sarcoma subtypes and an intense PPIX fluorescence was observed. PDT of CDXs resulted in a disruption of tumor-feeding vessels and 52.4% of CDXs presented as regressive after PDT treatment, whereas control CDXs remained vital in all cases. Therefore, 5-ALA mediated PDD and PDT appear to be promising tools in defining sarcoma resection margins (PDD) and adjuvant treatment of the tumor bed (PDT).
RESUMO
Synovial sarcoma, a rare malignant soft tissue tumor, is characterized by a specific chromosomal translocation t(X;18). The resulting chimeric SS18-SSX fusion protein drives synovial sarcoma pathogenesis by integrating into the BAF complex and dysregulating gene transcription. Because previous functional analyses revealed a connection between SS18-SSX and the activity of the transcriptional coregulators YAP1/TAZ and ß-catenin, respectively, this study examined a potential interdependence between these essential effector proteins in synovial sarcoma. In a large cohort of synovial sarcoma tissue specimens, IHC analyses revealed a substantial subset of synovial sarcoma with concurrent nuclear accumulation of YAP1/TAZ and ß-catenin. In vitro, small-molecule inhibitor treatment, RNAi-mediated knockdown, and vector-based overexpression assays demonstrated that YAP1, TAZ, and ß-catenin transcriptional activity is not only stimulated by the SS18-SSX fusion protein, but that they also mutually enhance each other's activation. These analyses showed the highest cooperative effect with overexpression of YAP1 in combination with ß-catenin. Coimmunoprecipitation experiments detected nuclear interactions between YAP1, ß-catenin, and the SS18-SSX fusion protein, the latter being an integral part of the BAF complex. Disruption of BAF complex assembly affected the coregulation of YAP1 and ß-catenin, indicating that this chromatin remodeling complex plays a crucial role for interdependent YAP1 and ß-catenin activation in synovial sarcoma cells. IMPLICATIONS: This study provides deeper insights into synovial sarcoma tumor biology demonstrating a mutual dependence between YAP1/TAZ and ß-catenin transcriptional activity and a complex interplay with the SS18-SSX fusion protein within the BAF complex.
Assuntos
Sarcoma Sinovial , beta Catenina , Humanos , beta Catenina/genética , Sarcoma Sinovial/genética , Sarcoma Sinovial/patologia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Núcleo Celular/metabolismoRESUMO
Ewing sarcoma (EwS) is a rare bone and soft tissue malignancy driven by chromosomal translocations encoding chimeric transcription factors, such as EWSR1-FLI1, that bind GGAA motifs forming novel enhancers that alter nearby expression. We propose that germline microsatellite variation at the 6p25.1 EwS susceptibility locus could impact downstream gene expression and EwS biology. We performed targeted long-read sequencing of EwS blood DNA to characterize variation and genomic features important for EWSR1-FLI1 binding. We identified 50 microsatellite alleles at 6p25.1 and observed that EwS-affected individuals had longer alleles (>135 bp) with more GGAA repeats. The 6p25.1 GGAA microsatellite showed chromatin features of an EWSR1-FLI1 enhancer and regulated expression of RREB1, a transcription factor associated with RAS/MAPK signaling. RREB1 knockdown reduced proliferation and clonogenic potential and reduced expression of cell cycle and DNA replication genes. Our integrative analysis at 6p25.1 details increased binding of longer GGAA microsatellite alleles with acquired EWSR-FLI1 to promote Ewing sarcomagenesis by RREB1-mediated proliferation.
Assuntos
Neoplasias Ósseas , Sarcoma de Ewing , Humanos , Alelos , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologiaRESUMO
Epstein-Barr virus (EBV) associated diffuse large B-cell lymphoma (DLBCL) represents a rare aggressive B-cell lymphoma subtype characterized by an adverse clinical outcome. EBV infection of lymphoma cells has been associated with different lymphoma subtypes while the precise role of EBV in lymphomagenesis and specific molecular characteristics of these lymphomas remain elusive. To further unravel the biology of EBV associated DLBCL, we present a comprehensive molecular analysis of overall 60 primary EBV positive (EBV+) DLBCLs using targeted sequencing of cancer candidate genes (CCGs) and genome-wide determination of recurrent somatic copy number alterations (SCNAs) in 46 cases, respectively. Applying the LymphGen classifier 2.0, we found that less than 20% of primary EBV + DLBCLs correspond to one of the established molecular DLBCL subtypes underscoring the unique biology of this entity. We have identified recurrent mutations activating the oncogenic JAK-STAT and NOTCH pathways as well as frequent amplifications of 9p24.1 contributing to immune escape by PD-L1 overexpression. Our findings enable further functional preclinical and clinical studies exploring the therapeutic potential of targeting these aberrations in patients with EBV + DLBCL to improve outcome.