Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

Pediatric BCOR-Altered Tumors From Soft Tissue/Kidney Display Specific DNA Methylation Profiles.

In the pediatric population, BCL6-correpresor gene (BCOR)-upregulated tumors include primitive myxoid mesenchymal tumors/undifferentiated sarcomas (PMMTI/UND), clear cell sarcomas of the kidney (CCSK), and high-grade neuroepithelial tumors (HG-NET). We investigated DNA methylation (DNAm) and copy number variation (CNV) profiling in these tumors (N = 34) using an Illumina EPIC BeadChip to better define the potential use of these tools to confirm diagnosis and predict outcomes. Twenty-seven tumors from 25 patients (age range, 0-10 years), showed molecular confirmation of genetic abnormalities as follows: BCOR internal tandem duplication in 14 PMMTI/UND, 8 CCSK, and 3 HG-NET and YWHAE fusions in 2 PMMTI/UND. The remaining 7 cases lacking informative molecular data were analyzed by immunophenotyping and were included in the study as a training cohort, clearly separated from the main study group. These were 4 PMMTI, 1 HG-NET, and 1 CCSK in which poor RNA preservation precluded the confirmation of BCOR rearrangements and 1 CCSK in which no rearrangements were found. DNAm data were compared with those of brain tumor and/or sarcoma classifier. Differentially methylated regions (DMRs) were analyzed in the 3 groups. Twenty-two cases of the 24 molecularly confirmed PMMTI/UND and CCSK and 3 of 6 of those with only immunophenotyping were classified within the methylation class "BCOR-altered sarcoma family" with optimal calibrated scores. PMMTI/UND and CCSK showed similar methylation profiles, whereas thousands of DMRs and significantly enriched pathways were evident between soft tissue/kidney tumors and HG-NET. The CNV analysis showed an overall flat profile in 19 of the 31 evaluable tumors (8/10 CCSK; 9/18 PMMTI/UND; 2/4 HG-NET). The most frequent CNVs were 1q gain and 9p and 10q loss. Follow-up time data were available for 20 patients: ≥2 CNV significantly correlated with a worse overall survival rate. In conclusion, soft tissue and kidney BCOR sarcomas matched with BCOR-altered sarcoma methylation class, whereas those from the brain matched with the central nervous system tumor classifier HG-NET BCOR, supporting the notion that DNAm profiling is an informative diagnostic tool. CNV alterations were associated with a more aggressive clinical behavior.

Proteomics of Aqueous Humor as a Source of Disease Biomarkers in Retinoblastoma.

Aqueous humor (AH) can be easily and safely used to evaluate disease-specific biomarkers in ocular disease. The aim of this study was to identify specific proteins biomarkers in the AH of retinoblastoma (RB) patients at various stages of the disease. We analyzed the proteome of 53 AH samples using high-resolution mass spectrometry. We grouped the samples according to active vitreous seeding (Group 1), active aqueous seeding (Group 2), naive RB (group 3), inactive RB (group 4), and congenital cataracts as the control (Group 5). We found a total of 889 proteins in all samples. Comparative parametric analyses among the different groups revealed three additional proteins expressed in the RB groups that were not expressed in the control group. These were histone H2B type 2-E (HISTH2B2E), InaD-like protein (PATJ), and ubiquitin conjugating enzyme E2 V1 (UBE2V1). Upon processing the data of our study with the OpenTarget Tool software, we found that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and CD44 were more highly expressed in the RB groups. Our results provide a proteome database regarding AH related to RB disease that may be used as a source of biomarkers. Further prospective studies should validate our finding in a large cohort of RB patients.

Radiogenomics prediction for MYCN amplification in neuroblastoma: A hypothesis generating study.

BACKGROUND: MYCN amplification represents a powerful prognostic factor in neuroblastoma (NB) and may occasionally account for intratumoral heterogeneity. Radiomics is an emerging field of advanced image analysis that aims to extract a large number of quantitative features from standard radiological images, providing valuable clinical information. PROCEDURE: In this retrospective study, we aimed to create a radiogenomics model by correlating computed tomography (CT) radiomics analysis with MYCN status. NB lesions were segmented on pretherapy CT scans and radiomics features subsequently extracted using a dedicated library. Dimensionality reduction/features selection approaches were then used for features procession and logistic regression models have been developed for the considered outcome. RESULTS: Seventy-eight patients were included in this study, as training dataset, of which 24 presented MYCN amplification. In total, 232 radiomics features were extracted. Eight features were selected through Boruta algorithm and two features were lastly chosen through Pearson correlation analysis: mean of voxel intensity histogram (p = .0082) and zone size non-uniformity (p = .038). Five-times repeated three-fold cross-validation logistic regression models yielded an area under the curve (AUC) value of 0.879 on the training set. The model was then applied to an independent validation cohort of 21 patients, of which five presented MYCN amplification. The validation of the model yielded a 0.813 AUC value, with 0.85 accuracy on previously unseen data. CONCLUSIONS: CT-based radiomics is able to predict MYCN amplification status in NB, paving the way to the in-depth analysis of imaging based biomarkers that could enhance outcomes prediction.

Tumour exosome integrins determine organotropic metastasis.

Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6ß4 and α6ß1 were associated with lung metastasis, while exosomal integrin αvß5 was linked to liver metastasis. Targeting the integrins α6ß4 and αvß5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

Establishment and Characterization of a Cell Line (S-RMS1) Derived from an Infantile Spindle Cell Rhabdomyosarcoma with SRF-NCOA2 Fusion Transcript.

Background: Spindle cell rhabdomyosarcoma (S-RMS) is a rare tumor that was previously considered as an uncommon variant of embryonal RMS (ERMS) and recently reclassified as a distinct RMS subtype with NCOA2, NCOA1, and VGLL2 fusion genes. In this study, we established a cell line (S-RMS1) derived from a four-month-old boy with infantile spindle cell RMS harboring SRF-NCOA2 gene fusion. Methods: Morphological and molecular characteristics of S-RMS1 were analyzed and compared with two RMS cell lines, RH30 and RD18. Whole genome sequencing of S-RMS1 and clinical exome sequencing of genomic DNA were performed. Results: S-RMS1 showed cells small in size, with a fibroblast-like morphology and positivity for MyoD-1, myogenin, desmin, and smooth muscle actin. The population doubling time was 3.7 days. Whole genome sequencing demonstrated that S-RMS1 retained the same genetic profile of the tumor at diagnosis. A Western blot analysis showed downregulation of AKT-p and YAP-p while RT-qPCR showed upregulation of endoglin and GATA6 as well as downregulation of TGFßR1 and Mef2C transcripts. Conclusion: This is the first report of the establishment of a cell line from an infantile spindle cell RMS with SRF-NCOA2 gene fusion. S-RMS1 should represent a useful tool for the molecular characterization of this rare and almost unknown tumor.

DNA Methylation Profiling for Diagnosing Undifferentiated Sarcoma with Capicua Transcriptional Receptor (CIC) Alterations.

Undifferentiated soft tissue sarcomas are a group of diagnostically challenging tumors in the pediatric population. Molecular techniques are instrumental for the categorization and differential diagnosis of these tumors. A subgroup of recently identified soft tissue sarcomas with undifferentiated round cell morphology was characterized by Capicua transcriptional receptor (CIC) rearrangements. Recently, an array-based DNA methylation analysis of undifferentiated tumors with small blue round cell histology was shown to provide a highly robust and reproducible approach for precisely classifying this diagnostically challenging group of tumors. We describe the case of an undifferentiated sarcoma of the abdominal wall in a 12-year-old girl. The patient presented with a voluminous mass of the abdominal wall, and multiple micro-nodules in the right lung. The tumor was unclassifiable with current immunohistochemical and molecular approaches. However, DNA methylation profiling allowed us to classify this neoplasia as small blue round cell tumor with CIC alterations. The patient was treated with neoadjuvant chemotherapy followed by complete surgical resection and adjuvant chemotherapy. After 22 months, the patient is disease-free and in good clinical condition. To put our experience in context, we conducted a literature review, analyzing current knowledge and state-of-the-art diagnosis, prognosis, and clinical management of CIC rearranged sarcomas. Our findings further support the use of DNA methylation profiling as an important tool to improve diagnosis of non-Ewing small round cell tumors.

Integration of Multiple Platforms for the Analysis of Multifluorescent Marking Technology Applied to Pediatric GBM and DIPG.

The intratumor heterogeneity represents one of the most difficult challenges for the development of effective therapies to treat pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG). These brain tumors are composed of heterogeneous cell subpopulations that coexist and cooperate to build a functional network responsible for their aggressive phenotype. Understanding the cellular and molecular mechanisms sustaining such network will be crucial for the identification of new therapeutic strategies. To study more in-depth these mechanisms, we sought to apply the Multifluorescent Marking Technology. We generated multifluorescent pGBM and DIPG bulk cell lines randomly expressing six different fluorescent proteins and from which we derived stable optical barcoded single cell-derived clones. In this study, we focused on the application of the Multifluorescent Marking Technology in 2D and 3D in vitro/ex vivo culture systems. We discuss how we integrated different multimodal fluorescence analysis platforms, identifying their strengths and limitations, to establish the tools that will enable further studies on the intratumor heterogeneity and interclonal interactions in pGBM and DIPG.

Exosomes in Systemic Sclerosis: Messengers Between Immune, Vascular and Fibrotic Components?

: Systemic sclerosis (SSc) is a rare autoimmune disease, characterized by vasculopathy and fibrosis of the skin and internal organs. This disease is still considered incurable and is associated with a high risk of mortality, which is related to fibrotic events. An early diagnosis is useful for preventing complications, and targeted therapies reduce disease progression and ameliorate patients' quality of life. Nevertheless, there are no validated biomarkers for early diagnosis with predictive prognostic value. Exosomes are membrane vesicles, transporting proteins and nucleic acids that may be delivered to target cells, which influences cellular behavior. They play important roles in cell-cell communication, both in physiological and pathological conditions, and may be useful as circulating biomarkers. Recent evidences suggest a role for these microvesicles in the three main aspects related to the pathogenesis of SSc (immunity, vascular damage, and fibrosis). Moreover, exosomes are of particular interest in the field of nano-delivery and are used as biological carriers. In this review, we report the latest information concerning SSc pathogenesis, clinical aspects of SSc, and current approaches to the treatment of SSc. Furthermore, we indicate a possible role of exosomes in SSc pathogenesis and suggest their potential use as diagnostic and prognostic biomarkers, as well as therapeutic tools.

Exosomal MiRNAs in Pediatric Cancers.

MicroRNAs (miRNAs) have generated great attention in oncology as they play a fundamental role in the regulation of gene expression and their aberrant expression is present in almost all types of tumors including pediatric ones. The discovery that miRNAs can be transported by exosomes, which are vesicles of 40-120 nm involved in cellular communication, that are produced by different cell types, and that are present in different biological fluids, has opened the possibility of using exosomal miRNAs as biomarkers. The possibility to diagnose and monitor the progression and response to drugs through molecules that can be easily isolated from biological fluids represents a particularly important aspect in the pediatric context where invasive techniques are often used. In recent years, the idea of liquid biopsy as well as studies on the possible role of exosomal miRNAs as biomarkers have developed greatly. In this review, we report an overview of all the evidences acquired in recent years on the identification of exosomal microRNAs with biomarker potential in pediatric cancers. We discuss the following herein: neuroblastoma, hepatoblastoma, sarcomas (osteosarcoma, Ewing's sarcoma and rhabdoid tumors, and non-rhabdomyosarcoma soft tissue sarcoma), brain tumors, lymphomas, and leukemias.

Evaluation of Endoglin (CD105) expression in pediatric rhabdomyosarcoma.

BACKGROUND: The Intratumoral Microvessel Density (IMVD) is commonly used to quantify tumoral vascularization and is usually assessed by pan-endothelial markers, such as CD31. Endoglin (CD105) is a protein predominantly expressed in proliferating endothelium and the IMVD determined by this marker measures specifically the neovascularization. In this study, we investigated the CD105 expression in pediatric rhabdomyosarcoma and assessed the neovascularization by using the angiogenic ratio IMVD-CD105 to IMVD-CD31. METHODS: Paraffin-embedded archival tumor specimens were selected from 65 pediatric patients affected by rhabdomyosarcoma. The expression levels of CD105, CD31 and Vascular Endothelial Growth Factor (VEGF) were investigated in 30 cases (18 embryonal and 12 alveolar) available for this study. The IMVD-CD105 to IMVD-CD31 expression ratio was correlated with clinical and pathologic features of these patients. RESULTS: We found a specific expression of endoglin (CD105) in endothelial cells of all the rhabdomyosarcoma specimens analyzed. We observed a significant positive correlation between the IMVD individually measured by CD105 and CD31. The CD105/CD31 expression ratio was significantly higher in patients with lower survival and embryonal histology. Indeed, patients with a CD105/CD31 expression ratio < 1.3 had a significantly increased OS (88%, 95%CI, 60%-97%) compared to patients with higher values (40%, 95%CI, 12%-67%). We did not find any statistical correlation among VEGF and EFS, OS and CD105/CD31 expression ratio. CONCLUSION: CD105 is expressed on endothelial cells of rhabdomyosarcoma and represent a useful tool to quantify neovascularization in this tumor. If confirmed by further studies, these results will indicate that CD105 is a potential target for combined therapies in rhabdomyosarcoma.

Congenital Rhabdomyosarcoma: a different clinical presentation in two cases.

BACKGROUND: Rhabdomyosarcoma (RMS), one of the most common soft tissue sarcomas of childhood, is very rare in the neonatal period (0.4-2% of cases). In order to gain a deeper understanding of this disease at such age, patient and tumor features, as well as treatment modality and outcome need to be reported. CASE PRESENTATION: We describe two cases with congenital RMS treated at Bambino Gesù Children's Hospital between 2000 and 2016. They represent only 2.24% of all RMS patients diagnosed during that period in our Institution; this data is in agreement with the incidence reported in the literature. They reflect the two different clinical forms in which the disease may manifest itself. One patient, with the alveolar subtype (positive for specific PAX3-FOXO1 fusion transcript) and disseminated disease, had a fatal outcome with central nervous system (CNS) progression despite conventional and high dose chemotherapy. The other child, with the localized embryonal subtype, was treated successfully with conservative surgery and conventional chemotherapy, including prolonged maintenance therapy. He is disease free at 7 years of follow-up. CONCLUSIONS: RMS can also be diagnosed during the neonatal period. Given the young age, disease management is often challenging, and especially for the alveolar subtype, the outcome is dismal despite intensified multimodality therapy. In fact, it characteristically manifests with multiple subcutaneous nodules and progression most commonly occurs in the CNS (Rodriguez-Galindo et al., Cancer 92(6):1613-20, 2001). In this context, CNS prophylaxis could play a role in preventing leptomeningeal dissemination, and molecular studies can allow a deeper tumor characterization, treatment stratification and identification of new potential therapeutic targets.

Proteomic Analysis of Neuroblastoma-Derived Exosomes: New Insights into a Metastatic Signature.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. Around 70% of patients with metastatic disease at diagnosis present bone-marrow infiltration, which is considered a marker of poor outcome; however, the mechanism underlying this specific tropism has to be elucidated. Tumor-derived exosomes may support metastatic progression in several tumors by interacting with the microenvironment, and may serve as tumor biomarkers. The main objective of this study is to identify an exosomal signature associated with NB metastatic bone-marrow dissemination. Therefore, the proteomic cargo of exosomes isolated from NB cell lines derived from primary tumor and bone-marrow metastasis is characterized. The comparison among exosomal proteins show 15 proteins exclusively present in primary tumor-derived exosomes, mainly involved in neuronal development, and 6 proteins in metastasis-derived exosomes related to cancer progression. Significant proteins obtain with statistical analysis performed between the two groups, reveal that primary tumor exosomes contain a higher level of proteins involved in extra-cellular matrix (ECM) assembly and adhesion, as well as in neuronal development. Exosomes isolated from bone-marrow metastasis exhibit proteins involved in ameboidal cell migration and mitochondrial activity. This work suggests that proteomic profiling of NB-derived exosomes reflects the tumor stage and may be considered as potential tumor biomarker.

Anomalous vascularization in a Wnt medulloblastoma: a case report.

BACKGROUND: Medulloblastoma is the most common malignant brain tumor in children. To date only few cases of medulloblastoma with hemorrhages have been reported in the literature. Although some studies speculate on the pathogenesis of this anomalous increased vascularization in medulloblastoma, the specific mechanism is still far from clearly understood. A correlation between molecular medulloblastoma subgroups and hemorrhagic features has not been reported, although recent preliminary studies described that WNT-subtype tumors display increased vascularization and hemorrhaging. CASE PRESENTATION: Herein, we describe a child with a Wnt-medulloblastoma presenting as cerebellar-vermian hemorrhagic lesion. Brain magnetic resonance imaging (MRI) showed the presence of a midline posterior fossa mass with a cystic hemorrhagic component. The differential diagnosis based on imaging included cavernous hemangioma, arteriovenous malformation and traumatic lesion. At surgery, the tumor appeared richly vascularized as documented by the preoperative angiography. CONCLUSIONS: The case we present showed that Wnt medulloblastoma may be associated with anomalous vascularization. Further studies are needed to elucidate if there is a link between the hypervascularization and the Wnt/ß-catenin signaling activation and if this abnormal vasculature might influence drug penetration contributing to good prognosis of this medulloblastoma subgroup.

Primary cutaneous and subcutaneous Ewing sarcoma.

BACKGROUND: Primary cutaneous/subcutaneous Ewing sarcoma (scEWS) is extremely rare. We describe clinical features, treatment, and outcome of this Ewing localization. PROCEDURE: Retrospective study (1996-2012) on 56 patients. RESULTS: Most primary scEWS occurred in late adolescent/young adult females (F/M = 1.9; median age 21.5 years), with primary tumor in the extremity/trunk (48.5%/39%). Only 35/56 samples had Real-Time-Polymerase-Chain-Reaction/Fluorescent-In-Situ-Hybridization analysis, 32/35 had EWS-translocation. Most of them exhibited known favorable prognostic factors: localized disease (54/56), initial tumor volume < 200 ml (51/53). Thirty and 25 patients received chemotherapy according to Euro-Ewing99 or a shorter/less intense chemotherapy regimen associated with milder toxicity. One patient had not received chemotherapy. Surgery was performed at diagnosis in 37 patients (18/37 marginal/intra-lesional resections) followed by secondary surgery in 8/37 (three remained marginal). Nineteen other patients had an initial biopsy followed by chemotherapy, 15/19 underwent late surgery (4/15 marginal/intra-lesional resections). Overall, 27/56 patients received radiotherapy. Median follow-up was six years (1-15). Two patients with metastatic disease progressed at metastatic sites. Four patients with localized disease experienced progression/relapse (local n = 3, metastatic n = 1). Survival was excellent: 5y-OS and 5y-EFS were 93.8% (95%CI = 83-98%) and 88.5% (95%CI-= 77-95), respectively. CONCLUSIONS: Unplanned primary surgery should be avoided to try to minimize potential long term sequels due to secondary surgery or radiotherapy. Biopsy with molecular analysis and staging should be performed at diagnosis to inform treatment recommendations. Patients with metastases should be treated aggressively as for other metastatic EWS. Further studies are necessary to clarify whether a less intensive chemotherapy regimen could be safely used in localized disease to minimize acute/late toxicities.

Patients in pediatric phase I and early phase II clinical oncology trials at Gustave Roussy: a 13-year center experience.

In the European Union, the pediatric medicines regulation in 2007 modified significantly the access to new agents in pediatric oncology. Early oncology trials are still thought to be associated with limited benefit and substantial risk. We report the characteristics and outcome of patients below 21 years enrolled in investigational trials in the Pediatric and Adolescent Department at Gustave Roussy between January 2000 and December 2012. A total of 235 patients (median age, 10.4 [0.8 to 20.7] y) were included in 26 trials (16 cytotoxic and 10 targeted agents) for a total of 260 inclusions. A total of 117 patients (50%) had brain tumors and 68 (29%) had various soft tissue and bone sarcoma. Thirteen of the 106 patients in a phase I trial experienced dose-limiting toxicity. Main severe toxicity was hematologic; none had toxic death. Grade 3 to 4 toxicities were associated with combination trials, cytotoxic agent, and at least 1 previous line of therapy. Thirty patients (12%) had objective response and 42 (16%) had stable disease for >4 months. Median overall survival was 9.0 months (95% CI, 7.5-10.5) and 73% of patients received further anticancer treatment. Phase I to II pediatric oncology trials are safe, associated with clinical benefit, and can be successfully integrated in current relapse strategies.

Natural history of cavernous malformations in children with brain tumors treated with radiotherapy and chemotherapy.

Cavernous malformations (CM) are cerebral irradiation-related late complications. Little is known about their natural history and the pathogenetic role of concomitant chemotherapy. We present a retrospective, single-institution study of 108 children affected with medulloblastoma, ependymoma, or germinoma treated with radio- and chemotherapy. The frequency, clinical and radiological presentations, and outcomes were analyzed to investigate the relationship among radiation dose, associated chemotherapy, age, latency and localization of radiation-induced CM. 100 out of 108 children were treated with radiotherapy for primary brain tumor; 34 (27 with medulloblastoma and 7 with other histologies) out of 100 patients developed CM. No significant relationship was found between CM and gender (p = 0.70), age (p = 0.90), use of specific chemotherapy (standard versus high-dose, p = 0.38), methotrexate (p = 0.49), and radiation dose (p = 0.45). However, CM developed more frequently and earlier when radiotherapy was associated with methotrexate (70 % of cases). Radiation-induced CM prevailingly occurred in the cerebral hemispheres (p = 0.0001). Only 3 patients (9 %) were symptomatic with headache. Three patients underwent surgery for intra- or extra-lesional hemorrhage. CM was confirmed by histopathology for all 3 patients. The vast majority of radiation-induced CM is asymptomatic, and macro-hemorrhagic events occur rarely. Concomitant therapy with methotrexate seems to favor their development. We recommend observation for asymptomatic lesions, while surgery should be reserved to symptomatic growth or hemorrhage.

Organoids as a new approach for improving pediatric cancer research.

A key challenge in cancer research is the meticulous development of models that faithfully emulates the intricacies of the patient scenario, with emphasis on preserving intra-tumoral heterogeneity and the dynamic milieu of the tumor microenvironment (TME). Organoids emerge as promising tool in new drug development, drug screening and precision medicine. Despite advances in the diagnoses and treatment of pediatric cancers, certain tumor subtypes persist in yielding unfavorable prognoses. Moreover, the prognosis for a significant portion of children experiencing disease relapse is dismal. To improve pediatric outcome many groups are focusing on the development of precision medicine approach. In this review, we summarize the current knowledge about using organoid system as model in preclinical and clinical solid-pediatric cancer. Since organoids retain the pivotal characteristics of primary parent tumors, they exert great potential in discovering novel tumor biomarkers, exploring drug-resistance mechanism and predicting tumor responses to chemotherapy, targeted therapy and immunotherapies. We also examine both the potential opportunities and existing challenges inherent organoids, hoping to point out the direction for future organoid development.

Plasma-derived extracellular vesicles miR-335-5p as potential diagnostic biomarkers for fusion-positive rhabdomyosarcoma.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma, with embryonal (ERMS) and alveolar (ARMS) representing the two most common histological subtypes. ARMS shows poor prognosis, being often metastatic at diagnosis. Thus, the discovery of novel biomarkers predictive of tumor aggressiveness represents one of the most important challenges to overcome and may help the development of tailored therapies. In the last years, miRNAs carried in extracellular vesicles (EVs), small vesicles of endocytic origin, have emerged as ideal candidate biomarkers due to their stability in plasma and their tissue specificity. METHODS: EVs miRNAs were isolated from plasma of 21 patients affected by RMS and 13 healthy childrens (HC). We performed a miRNA profile using the Serum/Plasma Focus microRNA PCR panels (Qiagen), and RT-qPCR for validation analysis. Statistically significant (p < 0.05) miRNAs were obtained by ANOVA test. RESULTS: We identified nine EVs miRNAs (miR-483-5p, miR-132-3p, miR-766-3p, miR-454-3p miR-197-3p, miR-335-3p, miR-17-5p, miR-486-5p and miR-484) highly upregulated in RMS patients compared to HCs. Interestingly, 4 miRNAs (miR-335-5p, miR-17-5p, miR-486-5p and miR-484) were significantly upregulated in ARMS samples compared to ERMS. In the validation analysis performed in a larger group of patients only three miRNAs (miR-483-5p, miR-335-5p and miR-484) were differentially significantly expressed in RMS patients compared to HC. Among these, mir-335-5p was significant also when compared ARMS to ERMS patients. MiR-335-5p was upregulated in RMS tumor tissues respect to normal tissues (p = 0.00202) and upregulated significantly between ARMS and ERMS (p = 0.04). Furthermore, the miRNA expression correlated with the Intergroup Rhabdomyosarcoma Study (IRS) grouping system, (p = 0.0234), and survival (OS, p = 0.044; PFS, p = 0.025). By performing in situ hybridization, we observed that miR-335-5p signal was exclusively in the cytoplasm of cancer cells. CONCLUSION: We identified miR-335-5p as significantly upregulated in plasma derived EVs and tumor tissue of patients affected by ARMS. Its expression correlates to stage and survival in patients. Future studies are needed to validate miR-335-5p as prognostic biomarker and to deeply elucidate its biological role.