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Osteosarcoma (OS) remains a dismal malignancy in children and young adults, with poor outcome for metastatic and recurrent disease. Immunotherapies in OS are not as promising as in some other cancer types due to intra-tumor heterogeneity and considerable off-target expression of the potentially targetable proteins. Here we show that chimeric antigen receptor (CAR) T cells could successfully target an isoform of alkaline phosphatase, ALPL-1, which is highly and specifically expressed in primary and metastatic OS. The target recognition element of the second-generation CAR construct is based on two antibodies, previously shown to react against OS. T cells transduced with these CAR constructs mediate efficient and effective cytotoxicity against ALPL-positive cells in in vitro settings and in state-of-the-art in vivo orthotopic models of primary and metastatic OS, without unexpected toxicities against hematopoietic stem cells or healthy tissues. In summary, CAR-T cells targeting ALPL-1 show efficiency and specificity in treating OS in preclinical models, paving the path for clinical translation., (© 2023. The Author(s).)

YAP1 and TAZ (WWTR1) oncoproteins are the final transducers of the Hippo tumor suppressor pathway. Deregulation of the pathway leads to YAP1/TAZ activation fostering tumorigenesis in multiple malignant tumor types, including sarcoma. However, oncogenic mutations within the core components of the Hippo pathway are uncommon. Ewing sarcoma (EwS), a pediatric cancer with low mutation rate, is characterized by a canonical fusion involving the gene EWSR1 and FLI1 as the most common partner. The fusion protein is a potent driver of oncogenesis, but secondary alterations are scarce, and little is known about other biological factors that determine the risk of relapse or progression. We have observed YAP1/TAZ expression and transcriptional activity in EwS cell lines. Analyses of 55 primary human EwS samples revealed that high YAP1/TAZ expression was associated with progression of the disease and predicted poorer outcome. We did not observe recurrent SNV or copy number gains/losses in Hippo pathway-related loci. However, differential CpG methylation of the RASSF1 locus (a regulator of the Hippo pathway) was observed in EwS cell lines compared with mesenchymal stem cells, the putative cell of origin of EwS. Hypermethylation of RASSF1 correlated with the transcriptional silencing of the tumor suppressor isoform RASFF1A, and transcriptional activation of the pro-tumorigenic isoform RASSF1C, which promotes YAP1/TAZ activation. Knockdown of YAP1/TAZ decreased proliferation and invasion abilities of EwS cells and revealed that YAP1/TAZ transcription activity is inversely correlated with the EWS-FLI1 transcriptional signature. This transcriptional antagonism could be explained partly by EWS-FLI1-mediated transcriptional repression of TAZ. Thus, YAP1/TAZ may override the transcriptional program induced by the fusion protein, contributing to the phenotypic plasticity determined by dynamic fluctuation of the fusion protein, a recently proposed model for disease dissemination in EwS. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Purpose: Goal of this study was to identify mechanisms that limit efficacy of trabectedin (ET-743, Yondelis) in Ewing sarcoma (EWS), so as to develop a clinical applicable combination therapy., Experimental Design: By chromatin immunoprecipitation, we analyzed EWS-FLI1 binding to the promoters of several target genes, such as TGFβR2, CD99, insulin-like growth factor receptor 1 (IGF1R), and IGF1, both in vitro and in xenografts treated with trabectedin or doxorubicin. Combined therapy with trabectedin and anti-IGF1R agents (AVE1642 HAb; OSI-906) was tested in vitro and in xenografts., Results: We confirm that both trabectedin and doxorubicin were able to strongly reduce EWS-FLI1 (both type I and type II) binding to two representative target genes (TGFβR2 and CD99), both in vitro and in xenografts. However, trabectedin, but not doxorubicin, was also able to increase the occupancy of EWS-FLI1 to IGF1R promoters, leading to IGF1R upregulation. Inhibition of IGF1R either by the specific AVE1642 human antibody or by the dual IGF1R/insulin receptor inhibitor OSI-906 (Linsitinib) greatly potentiate the efficacy of trabectedin in the 13 EWS cell lines here considered as well as in TC-71 and 6647 xenografts. Combined therapy induced synergistic cytotoxic effects. Trabectedin and OSI-906 deliver complementary messages that likely converge on DNA-damage response and repair pathways., Conclusions: We showed that trabectedin may not only inhibit but also enhance the binding of EWS-FLI1 to certain target genes, leading to upregulation of IGF1R. We here provide the rationale for combining trabectedin to anti-IGF1R inhibitors., (©2015 American Association for Cancer Research.)

Despite recent advances in the identification of the cytogenetic profiles of meningiomas, a significant group of tumors still show normal karyotypes or few chromosomal changes. The authors analyzed the cytogenetic profile of 50 meningiomas using fluorescence in situ hybridization and high-density (500 K) single nucleotide polymorphism (SNP) arrays. Our results confirm that del(22q) (52%) and del(1p) (16%) (common deleted regions: 22q11.21-22q13.3. and 1p31.2-p36.33) are the most frequent alterations. Additionally, recurrent monosomy 14 (8%), del(6q) (10%), del(7p) (10%), and del(19q) (4%) were observed, while copy number patterns consistent with recurrent chromosomal gains, gene amplification, and copy number neutral loss of heterozygosity (cnLOH) were either absent or rare. Based on their overall SNP profiles, meningiomas could be classified into: (i) diploid cases, (ii) meningiomas with a single chromosomal change [e.g., monosomy 22/del(22q)] and (iii) tumors with ≥2 altered chromosomes. In summary, our results confirm and extend on previous observations showing that the most recurrent chromosomal abnormalities in meningiomas correspond to chromosome losses localized in chromosomes 1, 22 and less frequently in chromosomes 6, 7, 14, and 19, while chromosomal gains and cnLOH are restricted to a small proportion of cases. Finally, a set of cancer-associated candidate genes associated with the TP53, MYC, CASP3, HDAC1, and TERT signaling pathways was identified, in cases with coexisting monosomy 14 and del(1p)., (Copyright © 2012 Wiley Periodicals, Inc.)

Adamantinomas of the long bones are low-grade malignant tumours. They seem to be related to osteofibrous dysplasia with a mesenchymal-to-epithelial transformation. We report a case of an adamantinoma with a revertant sarcomatoid transformation that showed a complete loss of epithelial differentiation. It corresponded to a 41-year-old male presented with an 8-cm multilobated lesion in the centre of the distal tibia. On the en bloc resection specimen, areas of classic adamantinoma were found but most of the tumor corresponded to a high-grade neoplasm with 2 histologic patterns: one made up by epithelial nests with a basaloid arrangement and positive for pankeratins and so-called glandular keratins, and a second sarcomatoid component, negative for these epithelial markers. Five months after surgery the patient had a massive relapse that consisted solely of the second sarcomatous component also negative for epithelial antibodies.Three cases of adamantinomas with sarcomatoid transformation of the epithelial component have been described but the tumours still preserved an epithelial immunophenotype. However, our case represents the extreme end of the sarcomatoid dedifferentiation of a classic adamantinoma with complete sarcomatoid transformation and total loss of epithelial markers. To our knowledge this has not been described previously.

Unlabelled: Treatment with fluororacil, epirubicin, and cyclophosphamide followed by weekly paclitaxel (FEC-P) yielded superior disease-free survival than FEC in the adjuvant breast cancer trial GEICAM 9906. We evaluate molecular subtypes predictive of prognosis and paclitaxel response in this trial. Two molecular subtype classifications based on conventional immunohistochemical and fluorescent in situ hybridization determinations were used: #1: Four groups segregated according to the combination of hormone receptor (HR) and HER2 status; #2: Intrinsic subtype classification (Triple Negative (TN), HER2, Luminal B and Luminal A)., Results: Both subtype classifications yielded prognostic and predictive information. HR +/HER2- patients (and Luminal A patients) had a significantly better outcome than the other subgroups of patients. The superiority of FEC-P over FEC was clearly more marked in HR-/HER2- patients (TN patients), particularly in the subset with basal phenotype (TN and either EGFR+ or cytokeratins 5/6+). The Luminal A subtype also achieved a significant benefit with FEC-P. The molecular-defined subgroup of TN was clearly predictive of better response to treatment with FEC-P. Luminal A patients had the best prognosis and also have a better outcome with weekly paclitaxel.

Background: Combinations of drug treatments based on bortezomib or lenalidomide plus steroids have resulted in very high response rates in multiple myeloma. However, most patients still relapse, indicating the need for novel combination partners to increase duration of response or to treat relapsed disease. We explored the antimyeloma activity of triple combinations of these well-established schemes with panobinostat, a novel deacetylase inhibitor with a multi-targeted profile., Design and Methods: The activity of these combinations was explored in vitro in cell lines by using MTT and annex-in V, ex vivo by flow cytometry, and in vivo using two different murine models of human myeloma: one bearing a subcutaneous plasmacytoma and another with a disseminated myeloma. Moreover, gene expression profiling and immunohistochemical studies were performed., Results: The addition of panobinostat (LBH589) to dexamethasone and either bortezomib or lenalidomide resulted in clear potentiation in multiple myeloma cell lines, freshly isolated plasma cells, and murine models of multiple myeloma. The quantification of the potency of these combinations by using the Chou-Talalay method showed synergistic combination indices for all of them. This effect derived from the deregulation of a cluster of genes that was completely different from the sum of genes affected by the single agents (895 and 1323 genes exclusively deregulated by panobinostat and dexamethasone plus bortezomib or lenalidomide, respectively). Functional experiments, such as annexin V staining, cell cycle analysis, and immunohistochemical studies also supported this potentiation. Anti-myeloma efficacy was confirmed in an extramedullary plasmacytoma model and a disseminated luciferized model, in which panobinostat also provided a marked benefit in bone disease., Conclusions: The potent activity, together with the exclusive mechanistic profile, provides the rationale for the clinical evaluation of these drug combinations in multiple myeloma.

Ewing's sarcoma family tumors (EFT) are characterized by specific chromosomal translocations, which lead to EWS/ETS transcription factors. Elucidation of EWS/ETS target gene networks within the context of other signaling pathways, together with the identification of the initiating cell, and the development of genetically engineered mice will hopefully lead to biology-based therapeutic strategies for these tumors.

Carcinosarcomas are malignant tumors with a mixture of carcinomatous and differentiated sarcomatous elements. We investigate the morphology, immunohistochemistry, and comparative genomic hybridization analysis of 3 mixed squamous carcinoma and osteosarcoma of the lung. All patients were male and their ages were 72, 43, and 58 years. The sizes of the neoplasms were 7, 5, and 5 cm in maximum diameter, respectively. Two patients died of the disease 9 and 14 months after surgery; and one is alive 6 months later. By light microscopy, all cases had both squamous and osteosarcomatous structures. Immunohistochemistry was positive for AE3AE1, p63, 34 E12, CAM 5.2 (2/3 cases), CK-7 (2/3 cases), epithelial membrane antigen, E-cadherin, p53, and carcinogenic embryonic antigen in carcinomatous areas, and for vimentin and CD-68 in sarcomatous component. Areas of transition positive for both cytokeratins and vimentin were seen in all cases. A total of 55 copy number changes were detected with a median of 18 abnormalities per case: 48 gains, 6 losses, and 1 high-level amplification. Chromosome alterations in osteosarcomatous areas were similar to those found in lung metastatic osteosarcoma, comparable to those found in carcinomatous areas and to lung squamous carcinomas. Coincidences between carcinomatous areas and osteosarcomatous zones were found as gains in chromosomes 1q, 3q, 5p, 8q, and 12p. These findings provide arguments that favor a common origin for both types of cells, supported by the mixture of cells, the existence of undifferentiated cells positive to both cytokeratin and vimentin markers, and the CGH overlaps of chromosomal gains between carcinomatous and sarcomatous areas.

Shugoshin-2 (SGOL2) is one of the two mammalian orthologs of the Shugoshin/Mei-S322 family of proteins that regulate sister chromatid cohesion by protecting the integrity of the multiprotein cohesin complexes. This protective system is essential for faithful chromosome segregation during mitosis and meiosis, which is the physical basis of Mendelian inheritance. Regardless of its evolutionary conservation from yeast to mammals, little is known about the in vivo relevance and specific role that SGOL2 plays in mammals. Here we show that disruption of the gene encoding mouse SGOL2 does not cause any alteration in sister chromatid cohesion in embryonic cultured fibroblasts and adult somatic tissues. Moreover, mutant mice develop normally and survive to adulthood without any apparent alteration. However, both male and female Sgol2-deficient mice are infertile. We demonstrate that SGOL2 is necessary for protecting centromeric cohesion during mammalian meiosis I. In vivo, the loss of SGOL2 promotes a premature release of the meiosis-specific REC8 cohesin complexes from anaphase I centromeres. This molecular alteration is manifested cytologically by the complete loss of centromere cohesion at metaphase II leading to single chromatids and physiologically with the formation of aneuploid gametes that give rise to infertility.

Background: Neuroblastic tumours (NBTs) represent a heterogeneous spectrum of neoplastic diseases associated with multiple genetic alterations. Structural and numerical chromosomal changes are frequent and are predictive parameters of NBTs outcome. We performed a comparative analysis of the biological entities constituted by NBTs with different ploidy status., Methods: Gene expression profiling of 49 diagnostic primary NBTs with ploidy data was performed using oligonucleotide microarray. Further analyses using Quantitative Real-Time Polymerase Chain Reaction (Q-PCR); array-Comparative Genomic Hybridization (aCGH); and Fluorescent in situ Hybridization (FISH) were performed to investigate the correlation between aneuploidy, chromosomal changes and gene expression profiles., Results: Gene expression profiling of 49 primary near-triploid and near-diploid/tetraploid NBTs revealed distinct expression profiles associated with each NBT subgroup. A statistically significant portion of genes mapped to 1p36 (P = 0.01) and 17p13-q21 (P < 0.0001), described as recurrently altered in NBTs. Over 90% of these genes showed higher expression in near-triploid NBTs and the majority are involved in cell differentiation pathways. Specific chromosomal abnormalities observed in NBTs, 1p loss, 17q and whole chromosome 17 gains, were reflected in the gene expression profiles. Comparison between gene copy number and expression levels suggests that differential expression might be only partly dependent on gene copy number. Intratumoural clonal heterogeneity was observed in all NBTs, with marked interclonal variability in near-diploid/tetraploid tumours., Conclusion: NBTs with different cellular DNA content display distinct transcriptional profiles with a significant portion of differentially expressed genes mapping to specific chromosomal regions known to be associated with outcome. Furthermore, our results demonstrate that these specific genetic abnormalities are highly heterogeneous in all NBTs, and suggest that NBTs with different ploidy status may result from different mechanisms of aneuploidy driving tumourigenesis.

Neuroblastoma (NB) is a pediatric neoplasia that shows complex combinations of acquired genetic aberrations. The specific genes and the molecular mechanisms responsible for development and progression of NB remain poorly understood. Our main objective is to compare the results obtained with different techniques for the detection of genomic data in 20 patients with NB using the information obtained to select the appropriate technique in routine analysis for the therapeutic stratification. The genetic methods used in this study are multiprobe fluorescence in situ hybridization (FISH) assay, metaphasic comparative genomic hybridization (mCGH), array comparative genomic hybridization (aCGH), and the multiplex ligation-dependent probe amplification (MLPA). Genomic copy number abnormalities were used to group the cases in four categories: MYCN amplification cases; 11q deletion tumors; cases with partial chromosome gains or losses and samples with entire chromosome alterations. The data obtained from the multigenomic techniques showed a high degree of concordance and our findings support the hypothesis that NB consists of biologically distinct subgroups that differ by genetic characteristics of prognostic relevance. FISH will be essential for the mandatory study of MYCN status. The use of MLPA as routine technique is an advantage procedure for detecting the implication of the common genetic alterations in NB.

A clear cell sarcoma-like tumor with osteoclast-like giant cells of the gastrointestinal tract without immunoexpression of CD117 was recently proposed as a new tumor entity. In this article, a case of a 37-year-old man with a neoplasm of the jejunum composed of polygonal cells with clear to eosinophilic cytoplasm forming nests and fascicles is reported. Giant cells were not identified. Immunohistochemically, the tumor cells expressed strongly S100 protein, human melanoma black 45, platelet-derived growth factor receptor alpha, B-cell lymphoma 2, p53, and to a lesser extent vimentin, neuron-specific enolase, and epithelial membrane antigen. Mindbomb homolog-1 index was 35%. Immunoreactivity for CD34 and CD117 was negative. The fluorescence in situ hybridization analysis showed a translocation involving chromosome 22q12, the diagnostic hallmark of clear cell sarcoma of soft tissues. This case indicates a close histogenetic relationship with the recently reported clear cell sarcoma with osteoclast-like giant cells of the gastrointestinal tract, as well as with the clear cell sarcoma of soft tissues.

Ewing sarcoma/primitive neuroectodermal tumors (EWS/PNET) are characterized by specific chromosomal translocations most often generating a chimeric EWS/FLI-1 gene. Depending on the number of juxtaposed exons assembled, several fusion types have been described with different incidences and prognoses. To assess the impact of each fusion type on the specific phenotypic, tumorigenic, and metastatic features of EWS/PNET, we developed an amenable system using a murine mesenchymal multipotent C3H10T1/2 cell line. Upon transduction of EWS/FLI-1, cells acquired dramatic morphological changes in vitro, including a smaller size and "neurite-like" membrane elongations. Chimeric fusion proteins conferred oncogenic properties in vitro, including anchorage-independent growth and an increased rate of proliferation. Furthermore, EWS/FLI-1 expression blocked mineralization, with concomitant repression of osteoblastic genes, and induced a dramatic repression of the adipocytic differentiation program. Moreover, EWS/FLI-1 promoted an aberrant neural phenotype by the de novo expression of specific neural genes. The intramuscular injection of transduced cells led to tumor development and the induction of overt osteolytic lesions. Analogously, to what was observed in human tumors, type 2 EWS/FLI-1 cells formed primary tumors in immunodeficient mice with a higher incidence and a lower latency than cells bearing types 1 and 3 fusions. By contrast, cells expressing types 2 and 3 fusions showed specific metastatic activity with a higher number of macroscopic metastases in soft tissues and osteolytic lesions in the limbs as compared to type-1-expressing cells. Therefore, the structure of each oncoprotein strongly influenced its tumorigenicity and metastagenicity. Thus, this model provides a basis for understanding the genetic determinants involved in Ewing tumor development and metastatic activity and represents a cellular system to analyze other oncoproteins involved in human sarcomagenesis.