Optical Genome Mapping versus Whole-Genome Sequencing in the Clinical Diagnosis of Gynecologic Mesenchymal Tumors Karin Wallander, Yingbo Lin, Vadym Ivanchuk, Valeria Difilippo, Venkatesh Chellappa, Sarath K. Murugan, Ingegerd Öfverholm, Robert Bränström, Karolin H. Nord, Joseph Carlson, Felix Haglund de Flon Journal of Molecular Diagnostics, 2026 Optical genome mapping (OGM) enables high-resolution detection of structural variants (SVs) and copy number aberrations (CNAs) using ultralong DNA molecules and minimal bioinformatics processing. Its diagnostic utility in solid tumors remains underexplored. Whole-genome sequencing (WGS) offers comprehensive variant detection but is resource intensive. This study presents a technical benchmarking of OGM versus WGS for mesenchymal tumors of the gynecologic tract. Twenty-five uterine mesenchymal tumors were prospectively analyzed using matched WGS, transcriptome sequencing, and OGM. Detected SVs, CNAs, and fusion genes were compared across platforms. OGM identified structural driver events in 80% of cases and demonstrated high concordance with WGS for major CNAs and translocations. In select cases, OGM resolved complex rearrangements not clearly defined by WGS, including a PLAG1::RERE fusion and an embedded inversion in a RAD51B::HMGA2 event. Conversely, WGS uniquely detected a truncating NF1 translocation and a TSC2::SENP3 fusion, both clinically significant. OGM is a technically robust platform for SV and CNA detection in mesenchymal tumors, and it may serve as an efficient alternative to sequencing-based cytogenomic approaches in selected clinical contexts, especially in tumors known to be driven by gross chromosomal rearrangements. WGS provides a comprehensive view of the cancer genome, suitable for tumors driven by single-nucleotide variants, SVs, and CNAs. The choice between platforms should be guided by clinical context, diagnostic needs, and available resources.
Mapping Genomic Heterogeneity in Pediatric and Adolescent–Young Adult Sarcomas: Insights from the Italian SAR-GEN2016 and SAR-GEN_ITA Prospective Multicenter Trials Elisa Tirtei, Valeria Difilippo, Federico Divincenzo, Sebastian Dorin Asaftei, Nicola Ratto, Raimondo Piana, Pietro Pellegrino, Alessandra Linari, Mauro Papotti, Katia Mareschi, Caterina Parlato, Simonetta Guarrera, Saverio Minucci, Marco Rabusin, Carla Manzitti, Arcangelo Prete, Federico Mercolini, Roberto Luksch, Cristina Meazza, Antonina Parafioriti, Angela Tamburini, Luca Coccoli, Rosamaria Mura, Marco Zecca, Emanuela Palmerini, Toni Ibrahim, Serena Peirone, Linda Penolazzi, Elvira De Luna, Celeste Cagnazzo, Sabrina Bombaci, Ivana Ferrero, Alessia Giovanna Santa Banche Niclot, Camilla Francesca Proto, Manuela Spadea, Paola Quarello, Elena Marini, Katiuscia Gizzi, Beatrice Fenoglio, Virginia Livellara, Alessandro Di Gangi, Nadia Puma, Giovanna Sironi, Andrea Di Bernardo, Matteo Cereda, Franca Fagioli Cancer Research Communications, 2026 Sarcomas in pediatric and adolescent–young adult (AYA) populations represent rare and biologically heterogeneous tumors with complex genetic underpinnings. Genomic profiling reveals subtype-specific alterations and therapeutic targets. Such tumors still represent an unmet clinical need due to limited treatment options and poorer outcomes, especially in advanced stages. Here, we present the SAR-GEN2016 and SAR-GEN_ITA clinical trials, conducted across 12 Italian centers, which enrolled 201 patients, including 158 bone and soft-tissue sarcoma samples collected at diagnosis or relapse. Whole-exome sequencing was successfully performed on 120 tumor samples. The most representative histotypes were osteosarcoma (n = 53), Ewing sarcoma (n = 39), rhabdomyosarcoma (n = 13), and synovial sarcoma (n = 5), and the genomic analyses were mainly focused on these subtypes. Overall, our cohort showed genomic differences between subtypes, highlighting how genomic complex sarcomas and fusion-driven sarcomas are distinct entities. The genomic complex histotypes, such as osteosarcoma, were characterized by a lower tumor mutational burden (TMB) and higher copy-number variation burden with enrichment of the CN2 signature. Recurrent and metastatic Ewing sarcomas have a higher TMB compared with treatment-naïve primary tumors, along with increased intratumoral heterogeneity. Oncogenic pathway analyses revealed dysregulation of the RTK–RAS and NOTCH pathways across subtypes, particularly in metastatic and recurrent tumors. In 71 of 120 analyzed samples (59%), at least one potentially actionable genomic alteration was identified, and 16% of those patients with relapsed disease received a matched targeted therapy based on the molecular profiling results. All findings were classified as ESCAT tier II or III. Our findings support the value of integrating genomic and clinical data to accelerate translational research in rare tumors. Significance: Pediatric and AYA sarcomas are rare with poor outcomes in advanced stages and limited treatment options. Through the SAR-GEN2016 and SAR-GEN_ITA multicenter trials, we performed whole-exome sequencing on 120 tumor samples with matched normal tissue from 158 patients with bone and soft-tissue sarcoma. Our integrative genomic analysis supports the genomic stratification and precision oncology in rare pediatric sarcomas.
The Role of RB1 and Secondary Genomic Changes in the Development of Spindle Cell and Pleomorphic Lipomas Maria Hellberg, Valeria Difilippo, Emilia Gottberg, Asle Hesla, Felix Haglund de Flon, Jenny Nilsson, Linda Magnusson, Saskia Sydow, Paul Piccinelli, Jakob Hofvander, Fredrik Mertens Genes Chromosomes and Cancer, 2025 Spindle cell lipomas (SCL) and pleomorphic lipomas (PL) are today considered a single tumor entity (SCLPL). Atypical SCLPL, in contrast, represents a recently recognized related but distinct entity. Here we explored the correlation between genomic features and morphological aspects of SCLPL and atypical SCLPL and the role of the RB1 gene in tumor development. Seventy‐one samples from 68 patients with SCLPL or atypical SCLPL, as well as a lipomatous tumor from a retinoblastoma patient with a germline pathogenic variant in the RB1 gene, and two pleomorphic liposarcomas were analyzed using chromosome banding, high‐resolution genomic arrays (SNP array), whole exome sequencing (WES), and/or RNA sequencing (RNA‐seq). Common for all tumors was involvement of 13q; other recurring variants were deletion of 16q, 6q, and 17p. A minimally deleted region that only contained RB1 was found on 13q. A distinction was seen between SCL on the one hand and PL and atypical SCLPL on the other; SCL had fewer copy number aberrations in general, and loss of 17p/ TP53 gene or a SNV affecting TP53 was only rarely detected in SCL but seen in the vast majority of PL and atypical SCLPL tumors. Thus, at the molecular level, SCL is different from PL/atypical SCLPL. Furthermore, the finding of the same copy number changes (loss of 13q, 6q, 16q, and 17p) in some pleomorphic liposarcomas raises the possibility that a subset of SCLPL/atypical SCLPL have the potential for malignant transformation.
CDK4 is co-amplified with either TP53 promoter gene fusions or MDM2 through distinct mechanisms in osteosarcoma Karim H. Saba, Valeria Difilippo, Emelie Styring, Jenny Nilsson, Linda Magnusson, Hilda van den Bos, René Wardenaar, Diana C. J. Spierings, Floris Foijer, Michaela Nathrath, Felix Haglund de Flon, Daniel Baumhoer, Karolin H. Nord Npj Genomic Medicine, 2024 Amplification of the MDM2 and CDK4 genes on chromosome 12 is commonly associated with low-grade osteosarcomas. In this study, we conducted high-resolution genomic and transcriptomic analyses on 33 samples from 25 osteosarcomas, encompassing both high- and low-grade cases with MDM2 and/or CDK4 amplification. We discerned four major subgroups, ranging from nearly intact genomes to heavily rearranged ones, each harbouring CDK4 and MDM2 amplification or CDK4 amplification with TP53 structural alterations. While amplicons involving MDM2 exhibited signs of an initial chromothripsis event, no evidence of chromothripsis was found in TP53-rearranged cases. Instead, the initial disruption of the TP53 locus led to co-amplification of the CDK4 locus. Additionally, we observed recurring promoter swapping events involving the regulatory regions of the FRS2, PLEKHA5, and TP53 genes. These events resulted in ectopic expression of partner genes, with the ELF1 gene being upregulated by the FRS2 and TP53 promoter regions in two distinct cases.
Osteosarcomas With Few Chromosomal Alterations or Adult Onset Are Genetically Heterogeneous Valeria Difilippo, Karim H. Saba, Emelie Styring, Linda Magnusson, Jenny Nilsson, Michaela Nathrath, Daniel Baumhoer, Karolin H. Nord Laboratory Investigation, 2024 Osteosarcoma is the most common primary bone malignancy, often detected in children and adolescents and commonly associated with TP53 alterations along with a high number of chromosomal rearrangements. However, osteosarcoma can affect patients of any age, and some tumors display less genetic complexity. Besides TP53 variants, data on key driving mutations are lacking for many osteosarcomas, particularly those affecting adults. To detect osteosarcoma-specific alterations, we screened transcriptomic and genomic sequencing and copy number data from 150 bone tumors originally diagnosed as osteosarcomas. To increase the precision in gene fusion detection, we developed a bioinformatic tool denoted as NAFuse, which extracts gene fusions that are verified at both the genomic and transcriptomic levels. Apart from the already reported genetic subgroups of osteosarcoma with TP53 structural variants, or MDM2 and/or CDK4 amplification, we did not identify any recurrent genetic driver that signifies the remaining cases. Among the plethora of mutations identified, we found genetic alterations characteristic of, or similar to, those of other bone and soft tissue tumors in 8 cases. These mutations were found in tumors with relatively few other genetic alterations or in adults. Due to the lack of clinical context and available tissue, we can question the diagnosis only on a genetic basis. However, our findings support the notion that osteosarcomas with few chromosomal alterations or adult onset seem genetically distinct from conventional osteosarcomas of children and adolescents.
CRAFT: A bioinformatics software for custom prediction of circular RNA functions Anna Dal Molin, Enrico Gaffo, Valeria Difilippo, Alessia Buratin, Caterina Tretti Parenzan, Silvia Bresolin, Stefania Bortoluzzi Briefings in Bioinformatics, 2022 Circular RNAs (circRNAs), transcripts generated by backsplicing, are particularly stable and pleiotropic molecules, whose dysregulation drives human diseases and cancer by modulating gene expression and signaling pathways. CircRNAs can regulate cellular processes by different mechanisms, including interaction with microRNAs (miRNAs) and RNA-binding proteins (RBP), and encoding specific peptides. The prediction of circRNA functions is instrumental to interpret their impact in diseases, and to prioritize circRNAs for functional investigation. Currently, circRNA functional predictions are provided by web databases that do not allow custom analyses, while self-standing circRNA prediction tools are mostly limited to predict only one type of function, mainly focusing on the miRNA sponge activity of circRNAs. To solve these issues, we developed CRAFT (CircRNA Function prediction Tool), a freely available computational pipeline that predicts circRNA sequence and molecular interactions with miRNAs and RBP, along with their coding potential. Analysis of a set of circRNAs with known functions has been used to appraise CRAFT predictions and to optimize its setting. CRAFT provides a comprehensive graphical visualization of the results, links to several knowledge databases, and extensive functional enrichment analysis. Moreover, it originally combines the predictions for different circRNAs. CRAFT is a useful tool to help the user explore the potential regulatory networks involving the circRNAs of interest and generate hypotheses about the cooperation of circRNAs into the modulation of biological processes.
