Biological Sample Collection to Advance Research and Treatment: A Fight Osteosarcoma Through European Research and Euro Ewing Consortium Statement.

Osteosarcoma and Ewing sarcoma are bone tumors mostly diagnosed in children, adolescents, and young adults. Despite multimodal therapy, morbidity is high and survival rates remain low, especially in the metastatic disease setting. Trials investigating targeted therapies and immunotherapies have not been groundbreaking. Better understanding of biological subgroups, the role of the tumor immune microenvironment, factors that promote metastasis, and clinical biomarkers of prognosis and drug response are required to make progress. A prerequisite to achieve desired success is a thorough, systematic, and clinically linked biological analysis of patient samples, but disease rarity and tissue processing challenges such as logistics and infrastructure have contributed to a lack of relevant samples for clinical care and research. There is a need for a Europe-wide framework to be implemented for the adequate and minimal sampling, processing, storage, and analysis of patient samples. Two international panels of scientists, clinicians, and patient and parent advocates have formed the Fight Osteosarcoma Through European Research consortium and the Euro Ewing Consortium. The consortia shared their expertise and institutional practices to formulate new guidelines. We report new reference standards for adequate and minimally required sampling (time points, diagnostic samples, and liquid biopsy tubes), handling, and biobanking to enable advanced biological studies in bone sarcoma. We describe standards for analysis and annotation to drive collaboration and data harmonization with practical, legal, and ethical considerations. This position paper provides comprehensive guidelines that should become the new standards of care that will accelerate scientific progress, promote collaboration, and improve outcomes.

(B)On(e)-cohistones and the epigenetic alterations at the root of bone cancer.

Identification of mutations in histones in a number of human neoplasms and developmental syndromes represents the most compelling evidence to date for a causal role of epigenetic perturbations in human disease. In most cases, these mutations have gain of function properties that cause deviation from normal developmental processes leading to embryo defects and/or neoplastic transformation. These exciting discoveries represent a step-change in our understanding of the role of chromatin (dys)regulation in development and disease. However, the mechanisms of action of oncogenic histone mutations (oncohistones) remain only partially understood. Here, we critically assess existing literature on oncohistones focussing mainly on bone neoplasms. We show how it is possible to draw parallels with some of the cell-autonomous mechanisms of action described in paediatric brain cancer, although the functions of oncohistones in bone tumours remain under-investigated. In this respect, it is becoming clear that histone mutations targeting the same residues display, at least in part, tissue-specific oncogenic mechanisms. Furthermore, it is emerging that cancer cells carrying oncohistones can modify the surrounding microenvironment to support growth and/or alter differentiation trajectories. A better understanding of oncohistone function in different neoplasms provide potential for identification of signalling that could be targeted therapeutically. Finally, we discuss some of the main concepts and future directions in this research area, while also drawing possible connections and parallels with other cancer epigenetic mechanisms.

Aberrant paracrine signalling for bone remodelling underlies the mutant histone-driven giant cell tumour of bone.

Oncohistones represent compelling evidence for a causative role of epigenetic perturbations in cancer. Giant cell tumours of bone (GCTs) are characterised by a mutated histone H3.3 as the sole genetic driver present in bone-forming osteoprogenitor cells but absent from abnormally large bone-resorbing osteoclasts which represent the hallmark of these neoplasms. While these striking features imply a pathogenic interaction between mesenchymal and myelomonocytic lineages during GCT development, the underlying mechanisms remain unknown. We show that the changes in the transcriptome and epigenome in the mesenchymal cells caused by the H3.3-G34W mutation contribute to increase osteoclast recruitment in part via reduced expression of the TGFß-like soluble factor, SCUBE3. Transcriptional changes in SCUBE3 are associated with altered histone marks and H3.3G34W enrichment at its enhancer regions. In turn, osteoclasts secrete unregulated amounts of SEMA4D which enhances proliferation of mutated osteoprogenitors arresting their maturation. These findings provide a mechanism by which GCTs undergo differentiation in response to denosumab, a drug that depletes the tumour of osteoclasts. In contrast, hTERT alterations, commonly found in malignant GCT, result in the histone-mutated neoplastic cells being independent of osteoclasts for their proliferation, predicting unresponsiveness to denosumab. We provide a mechanism for the initiation of GCT, the basis of which is dysfunctional cross-talk between bone-forming and bone-resorbing cells. The findings highlight the role of tumour/microenvironment bidirectional interactions in tumorigenesis and how this is exploited in the treatment of GCT.

Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants.

CRISPR/Cas9, base editors and prime editors comprise the contemporary genome editing toolbox. Many studies have optimized the use of CRISPR/Cas9, as the original CRISPR genome editing system, in substituting single nucleotides by homology directed repair (HDR), although this remains challenging. Studies describing modifications that improve editing efficiency fall short of isolating clonal cell lines or have not been validated for challenging loci or cell models. We present data from 95 transfections using a colony forming and an immortalized cell line comparing the effect on editing efficiency of donor template modifications, concentration of components, HDR enhancing agents and cold shock. We found that in silico predictions of guide RNA efficiency correlated poorly withactivity in cells. Using NGS and ddPCR we detected editing efficiencies of 5-12% in the transfected populations which fell to 1% on clonal cell line isolation. Our data demonstrate the variability of CRISPR efficiency by cell model, target locus and other factors. Successful genome editing requires a comparison of systems and modifications to develop the optimal protocol for the cell model and locus. We describe the steps in this process in a flowchart for those embarking on genome editing using any system and incorporate validated HDR-boosting modifications for those using CRISPR/Cas9.

AZD8055 enhances in vivo efficacy of afatinib in chordomas.

Chordomas are primary bone tumors that arise in the cranial base, mobile spine, and sacrococcygeal region, affecting patients of all ages. Currently, there are no approved agents for chordoma patients. Here, we evaluated the anti-tumor efficacy of small molecule inhibitors that target oncogenic pathways in chordoma, as single agents and in combination, to identify novel therapeutic approaches with the greatest translational potential. A panel of small molecule compounds was screened in vivo against patient-derived xenograft (PDX) models of chordoma, and potentially synergistic combinations were further evaluated using chordoma cell lines and xenograft models. Among the tested agents, inhibitors of EGFR (BIBX 1382, erlotinib, and afatinib), c-MET (crizotinib), and mTOR (AZD8055) significantly inhibited tumor growth in vivo but did not induce tumor regression. Co-inhibition of EGFR and c-MET using erlotinib and crizotinib synergistically reduced cell viability in chordoma cell lines but did not result in enhanced in vivo activity. Co-inhibition of EGFR and mTOR pathways using afatinib and AZD8055 synergistically reduced cell viability in chordoma cell lines. Importantly, this dual inhibition completely suppressed tumor growth in vivo, showing improved tumor control. Together, these data demonstrate that individual inhibitors of EGFR, c-MET, and mTOR pathways suppress chordoma growth both in vitro and in vivo. mTOR inhibition increased the efficacy of EGFR inhibition on chordoma growth in several preclinical models. The insights gained from our study potentially provide a novel combination therapeutic strategy for patients with chordoma. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Inhibition of Histone H3K27 Demethylases Inactivates Brachyury (TBXT) and Promotes Chordoma Cell Death.

Expression of the transcription factor brachyury (TBXT) is normally restricted to the embryo, and its silencing is epigenetically regulated. TBXT promotes mesenchymal transition in a subset of common carcinomas, and in chordoma, a rare cancer showing notochordal differentiation, TBXT acts as a putative oncogene. We hypothesized that TBXT expression is controlled through epigenetic inhibition to promote chordoma cell death. Screening of five human chordoma cell lines revealed that pharmacologic inhibition of the histone 3 lysine 27 demethylases KDM6A (UTX) and KDM6B (JMJD3) leads to cell death. This effect was phenocopied by dual genetic inactivation of KDM6A/B using CRISPR/Cas9. Inhibition of KDM6A/B with a novel compound KDOBA67 led to a genome-wide increase in repressive H3K27me3 marks with concomitant reduction in active H3K27ac, H3K9ac, and H3K4me3 marks. TBXT was a KDM6A/B target gene, and chromatin changes at TBXT following KDOBA67 treatment were associated with a reduction in TBXT protein levels in all models tested, including primary patient-derived cultures. In all models tested, KDOBA67 treatment downregulated expression of a network of transcription factors critical for chordoma survival and upregulated pathways dominated by ATF4-driven stress and proapoptotic responses. Blocking the AFT4 stress response did not prevent suppression of TBXT and induction of cell death, but ectopic overexpression of TBXT increased viability, therefore implicating TBXT as a potential therapeutic target of H3K27 demethylase inhibitors in chordoma. Our work highlights how knowledge of normal processes in fetal development can provide insight into tumorigenesis and identify novel therapeutic approaches. SIGNIFICANCE: Pharmacologic inhibition of H3K27-demethylases in human chordoma cells promotes epigenetic silencing of oncogenic TBXT, alters gene networks critical to survival, and represents a potential novel therapy.

Frequent alterations in p16/CDKN2A identified by immunohistochemistry and FISH in chordoma.

The expression of p16/CDKN2A, the second most commonly inactivated tumour suppressor gene in cancer, is lost in the majority of chordomas. However, the mechanism(s) leading to its inactivation and contribution to disease progression have only been partially addressed using small patient cohorts. We studied 384 chordoma samples from 320 patients by immunohistochemistry and found that p16 protein was lost in 53% of chordomas and was heterogeneously expressed in these tumours. To determine if CDKN2A copy number loss could explain the absence of p16 protein expression we performed fluorescence in situ hybridisation (FISH) for CDKN2A on consecutive tissue sections. CDKN2A copy number status was altered in 168 of 274 (61%) of samples and copy number loss was the most frequent alteration acquired during clinical disease progression. CDKN2A homozygous deletion was always associated with p16 protein loss but only accounted for 33% of the p16-negative cases. The remaining immunonegative cases were associated with disomy (27%), monosomy (12%), heterozygous loss (20%) and copy number gain (7%) of CDKN2A, supporting the hypothesis that loss of protein expression might be achieved via epigenetic or post-transcriptional regulatory mechanisms. We identified that mRNA levels were comparable in tumours with and without p16 protein expression, but other events including DNA promoter hypermethylation, copy number neutral loss of heterozygosity and expression of candidate microRNAs previously implicated in the regulation of CDKN2A expression were not identified to explain the protein loss. The data argue that p16 loss in chordoma is commonly caused by a post-transcriptional regulatory mechanism that is yet to be defined.

Synovial chondromatosis and soft tissue chondroma: extraosseous cartilaginous tumor defined by FN1 gene rearrangement.

A fusion between fibronectin 1 (FN1) and activin receptor 2A (ACVR2A) has been reported previously in isolated cases of the synovial chondromatosis. To analyze further and validate the findings, we performed FISH and demonstrated recurrent FN1-ACVR2A rearrangements in synovial chondromatosis (57%), and chondrosarcoma secondary to synovial chondromatosis (75%), showing that FN1 and/or AVCR2A gene rearrangements do not distinguish between benign and malignant synovial chondromatosis. RNA sequencing revealed the presence of the FN1-ACVR2A fusion in several cases that were negative by FISH suggesting that the true prevalence of this fusion is potentially higher than 57%. In soft tissue chondromas, FN1 alterations were detected by FISH in 50% of cases but no ACVR2A alterations were identified. RNA sequencing identified a fusion involving FN1 and fibroblast growth factor receptor 2 (FGFR2) in the case of soft tissue chondroma and FISH confirmed recurrent involvement of both FGFR1 and FGFR2. These fusions were present in a subset of soft tissue chondromas characterized by grungy calcification, a feature reminiscent of phosphaturic mesenchymal tumor. However, unlike the latter, fibroblast growth factor 23 (FGF23) mRNA expression was not elevated in soft tissue chondromas harboring the FN1-FGFR1 fusion. The mutual exclusivity of ACVR2A rearrangements observed in synovial chondromatosis and FGFR1/2 in soft tissue chondromas suggests these represent separate entities. There have been no reports of malignant soft tissue chondromas, therefore differentiating these lesions will potentially alter clinical management by allowing soft tissue chondromas to be managed more conservatively.

The peritoneum: healing, immunity, and diseases.

The peritoneum defines a confined microenvironment, which is stable under normal conditions, but is exposed to the damaging effect of infections, surgical injuries, and other neoplastic and non-neoplastic events. Its response to damage includes the recruitment, proliferation, and activation of a variety of haematopoietic and stromal cells. In physiological conditions, effective responses to injuries are organized; inflammatory triggers are eliminated; inflammation quickly abates; and the normal tissue architecture is restored. However, if inflammatory triggers are not cleared, fibrosis or scarring occurs and impaired tissue function ultimately leads to organ failure. Autoimmune serositis is characterized by the persistence of self-antigens and a relapsing clinical pattern. Peritoneal carcinomatosis and endometriosis are characterized by the persistence of cancer cells or ectopic endometrial cells in the peritoneal cavity. Some of the molecular signals orchestrating the recruitment of inflammatory cells in the peritoneum have been identified in the last few years. Alternative activation of peritoneal macrophages was shown to guide angiogenesis and fibrosis, and could represent a novel target for molecular intervention. This review summarizes current knowledge of the alterations to the immune response in the peritoneal environment, highlighting the ambiguous role played by persistently activated reparative macrophages in the pathogenesis of common human diseases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Leukocytes recruited by tumor-derived HMGB1 sustain peritoneal carcinomatosis.

The factors that determine whether disseminated transformed cells in vivo yield neoplastic lesions have only been partially identified. We established an ad hoc model of peritoneal carcinomatosis by injecting colon carcinoma cells in mice. Tumor cells recruit inflammatory leukocytes, mostly macrophages, and generate neoplastic peritoneal lesions. Phagocyte depletion via clodronate treatment reduces neoplastic growth. Colon carcinoma cells release a prototypic damage-associated molecular pattern (DAMP)/alarmin, High Mobility Group Box1 (HMGB1), which attracts leukocytes. Exogenous HMGB1 accelerates leukocyte recruitment, macrophage infiltration, tumor growth and vascularization. Lentiviral-based HMGB1 knockdown or pharmacological interference with its extracellular impair macrophage recruitment and tumor growth. Our findings provide a preclinical proof of principle that strategies based on preventing HMGB1-driven recruitment of leukocytes could be used for treating peritoneal carcinomatosis.

EGFR inhibitors identified as a potential treatment for chordoma in a focused compound screen.

Chordoma is a rare malignant bone tumour with a poor prognosis and limited therapeutic options. We undertook a focused compound screen (FCS) against 1097 compounds on three well-characterized chordoma cell lines; 154 compounds were selected from the single concentration screen (1 µm), based on their growth-inhibitory effect. Their half-maximal effective concentration (EC50 ) values were determined in chordoma cells and normal fibroblasts. Twenty-seven of these compounds displayed chordoma selective cell kill and 21/27 (78%) were found to be EGFR/ERBB family inhibitors. EGFR inhibitors in clinical development were then studied on an extended cell line panel of seven chordoma cell lines, four of which were sensitive to EGFR inhibition. Sapitinib (AstraZeneca) emerged as the lead compound, followed by gefitinib (AstraZeneca) and erlotinib (Roche/Genentech). The compounds were shown to induce apoptosis in the sensitive cell lines and suppressed phospho-EGFR and its downstream pathways in a dose-dependent manner. Analysis of substituent patterns suggested that EGFR-inhibitors with small aniline substituents in the 4-position of the quinazoline ring were more effective than inhibitors with large substituents in that position. Sapitinib showed significantly reduced tumour growth in two xenograft mouse models (U-CH1 xenograft and a patient-derived xenograft, SF8894). One of the resistant cell lines (U-CH2) was shown to express high levels of phospho-MET, a known bypass signalling pathway to EGFR. Neither amplifications (EGFR, ERBB2, MET) nor mutations in EGFR, ERBB2, ERBB4, PIK3CA, BRAF, NRAS, KRAS, PTEN, MET or other cancer gene hotspots were detected in the cell lines. Our findings are consistent with the reported (p-)EGFR expression in the majority of clinical samples, and provide evidence for exploring the efficacy of EGFR inhibitors in the treatment of patients with chordoma and studying possible resistance mechanisms to these compounds in vitro and in vivo. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

5-Fluorouracil causes leukocytes attraction in the peritoneal cavity by activating autophagy and HMGB1 release in colon carcinoma cells.

Signals released by leukocytes contribute to tumor growth and influence the efficacy of antineoplastic treatments. The outcome of peritoneal carcinomatosis treatments is unsatisfactory, possibly because chemotherapy activates events that have in the long-term deleterious effects. In this study we offer evidence that 5-fluorouracile (5-FU), besides provoking apoptosis of MC38 colon carcinoma cells, induces a striking attraction of leukocytes both in an orthotopic model of colon carcinomatosis in vivo and in monocyte-migration assays in vitro. Leukocyte attraction depends on the presence of High Mobility Group Box 1 (HMGB1), an endogenous immune adjuvant and chemoattractant released by dying cells. Leukocyte recruitment is prevented in vivo and in vitro using blocking antibodies against HMGB1 and its competitive antagonist BoxA or by interfering with HMGB1 expression. Autophagy is required for leukocyte chemoattraction, since the latter abates upon pharmacological blockade of the autophagic flux while activation of autophagy per se, in the absence of death of colon carcinoma cells, is not sufficient to attract leukocytes. Our results identify autophagy induction and HMGB1 release in colon carcinoma cells as key events responsible for 5-FU elicited leukocyte attraction and define a novel rate-limiting target for combinatorial therapies.

Cathepsin C is a tissue-specific regulator of squamous carcinogenesis.

Serine and cysteine cathepsin (Cts) proteases are an important class of intracellular and pericellular enzymes mediating multiple aspects of tumor development. Emblematic of these is CtsB, reported to play functionally significant roles during pancreatic islet and mammary carcinogenesis. CtsC, on the other hand, while up-regulated during pancreatic islet carcinogenesis, lacks functional significance in mediating neoplastic progression in that organ. Given that protein expression and enzymatic activity of both CtsB and CtsC are increased in numerous tumors, we sought to understand how tissue specificity might factor into their functional significance. Thus, whereas others have reported that CtsB regulates metastasis of mammary carcinomas, we found that development of squamous carcinomas occurs independently of CtsB. In contrast to these findings, our studies found no significant role for CtsC during mammary carcinogenesis but revealed squamous carcinogenesis to be functionally dependent on CtsC. In this context, dermal/stromal fibroblasts and bone marrow-derived cells expressed increased levels of enzymatically active CtsC that regulated the complexity of infiltrating immune cells in neoplastic skin, development of angiogenic vasculature, and overt squamous cell carcinoma growth. These studies highlight the important contribution of tissue/microenvironment context to solid tumor development and indicate that tissue specificity defines functional significance for these two members of the cysteine protease family.

The nitric oxide-donor molsidomine modulates the innate inflammatory response in a mouse model of muscular dystrophy.

Inflammation plays a crucial role in muscle remodeling and repair after acute and chronic damage, in particular in muscular dystrophies, a heterogeneous group of genetic diseases leading to muscular degeneration. Defect of nitric oxide (NO) generation is a key pathogenic event in muscular dystrophies, thus NO donors have been explored as new therapeutics for this disease. We have investigated the immune-modulating effect of one of such drugs, molsidomine, able to slow the progression of muscular dystrophy in the α-Sarcoglican-null mice, a model for the limb girdle muscular dystrophy 2D, sharing several hallmarks of muscle degeneration with other muscular dystrophies. α-Sarcoglican-null mice were treated with molsidomine and drug effects on the inflammatory infiltrates and on muscle repair were assessed at selected time points. We found that molsidomine treatment modulates effectively the characteristics of the inflammatory infiltrate within dystrophic muscles, enhancing its healing function. Initially molsidomine amplified macrophage recruitment, promoting a more efficient clearance of cell debris and effective tissue regeneration. At a later stage molsidomine decreased significantly the extent of the inflammatory infiltrate, whose persistence exacerbates muscle damage: most of the remaining macrophages displayed characteristics of the transitional population, associated with reduced fibrosis and increased preservation of the muscle tissue. The dual action of molsidomine, the already known NO donation and the immunomodulatory function we now identified, suggests that it has a unique potential in tissue healing during chronic muscle damage. This, alongside its already approved use in human, makes molsidomine a drug with a significant therapeutic potential in muscular dystrophies.

Evaluation of the role of tumor-associated macrophages in an experimental model of peritoneal carcinomatosis using (18)F-FDG PET.

UNLABELLED: PET is widely used at the clinical and preclinical levels for tumor assessment and evaluation of treatment efficacy. Here, we established and took advantage of a preclinical model of peritoneal carcinomatosis to evaluate the contribution of inflammatory infiltrating macrophages in tumor progression that was followed using (18)F-FDG PET. METHODS: Groups of mice with peritoneal carcinomatosis were longitudinally evaluated with (18)F-FDG PET. Intraperitoneal depletion of macrophages was achieved by an approach (i.e., administration of clodronate encapsulated into liposomes) that proved to be safe and effective. Sham liposomes were used in control animal cohorts. RESULTS: (18)F-FDG PET allowed us to detect and monitor peritoneal lesion growth and diffusion. Macrophage-depleted animals showed a substantial reduction in tumor burden paralleled by a decrement in the extent of radioactivity distribution. A significant correlation between lesion dimension and metabolic volume was observed not only in macrophage-depleted but also in sham-treated mice. CONCLUSION: (18)F-FDG PET allowed a noninvasive detection of peritoneal carcinomatosis lesions. Although macrophages play a key role in the early growth and spreading of lesions in the peritoneal cavity, neoplastic cells apparently represent the major player in this system in the uptake of (18)F-FDG.

Proangiogenic Tie2(+) macrophages infiltrate human and murine endometriotic lesions and dictate their growth in a mouse model of the disease.

Endometriosis affects women of reproductive age, causing infertility and pain. Although immune cells are recruited in endometriotic lesions, their role is unclear. Tie2-expressing macrophages (TEMs) have nonredundant functions in promoting angiogenesis and growth of experimental tumors. Here we show that human TEMs infiltrate areas surrounding newly formed endometriotic blood vessels. We set up an ad hoc mouse model in which TEMs, and not Tie2-expressing endothelial cells, are targeted. We transplanted in wild-type recipients bone marrow cells expressing a suicide gene (Herpes simplex virus type 1 thymidine kinase) under the Tie2 promoter/enhancer. TEMs infiltrated endometriotic lesions. TEM depletion by ganciclovir administration arrested the growth of established lesions, without toxicity. Lesion architecture was disrupted, with: i) loss of glandular organization, ii) reduced neovascularization, and iii) activation of caspase 3 in CD31(+) endothelial cells. Thus, TEMs are important for maintaining the viability of newly formed vessels and represent a potential therapeutic target in endometriosis.

Macrophages are alternatively activated in patients with endometriosis and required for growth and vascularization of lesions in a mouse model of disease.

The mechanisms that sustain endometrial tissues at ectopic sites in patients with endometriosis are poorly understood. Various leukocytes, including macrophages, infiltrate endometriotic lesions. In this study, we depleted mouse macrophages by means of either clodronate liposomes or monoclonal antibodies before the injection of syngeneic endometrial tissue. In the absence of macrophages, tissue fragments adhered and implanted into the peritoneal wall, but endometriotic lesions failed to organize and develop. When we depleted macrophages after the establishment of endometriotic lesions, blood vessels failed to reach the inner layers of the lesions, which stopped growing. Macrophages from patients with endometriosis and experimental mice, but not nonendometriotic patients who underwent surgery for uterine leiomyomas or control mice, expressed markers of alternative activation. These markers included high levels of scavenger receptors, CD163 and CD206, which are involved in both the scavenging of hemoglobin with iron transfer into macrophages and the silent clearance of inflammatory molecules. Macrophages in both inflammatory liquid and ectopic lesions were equally polarized, suggesting a critical role of environmental cues in the peritoneal cavity. Adoptively transferred, alternatively activated macrophages dramatically enhanced endometriotic lesion growth in mice. Inflammatory macrophages effectively protected mice from endometriosis. Therefore, endogenous macrophages involved in tissue remodeling appear as players in the natural history of endometriosis, required for effective vascularization and ectopic lesion growth.