Ewing sarcomas (ES) are rare small round cell sarcomas often affecting children and characterized by gene fusions involving one member of the FET family of genes (usually EWSR1) and a member of the ETS family of transcription factors (usually FLI1 or ERG). The detection of EWSR1 rearrangements has important diagnostic value. Here, we conducted a retrospective review of 218 consecutive pediatric ES at diagnosis and found eight patients having data from chromosome analysis, FISH/microarray, and gene-fusion assay. Three of these eight ES had novel complex/cryptic EWSR1 rearrangements/fusions by chromosome analysis. One case had a t(9;11;22)(q22;q24;q12) three-way translocation involving EWSR1::FLI1 fusion and 1q jumping translocation. Two cases had cryptic EWSR1 rearrangements/fusions, including one case with a cryptic t(4;11;22)(q35;q24;q12) three-way translocation involving EWSR1::FLI1 fusion, and the other had a cryptic EWSR1::ERG rearrangement/fusion on an abnormal chromosome 22. All patients in this study had various aneuploidies with a gain of chromosome 8 (75%), the most common, followed by a gain of chromosomes 20 (50%) and 4 (37.5%), respectively. Recognition of complex and/or cryptic EWSR1 gene rearrangements/fusions and other chromosome abnormalities (such as jumping translocation and aneuploidies) using a combination of various genetic methods is important for accurate diagnosis, prognosis, and treatment outcomes of pediatric ES.

Chromoanagenesis is a catastrophic genomic phenomenon involving sudden, extensive rearrangements within one or a few cell cycles. In osteosarcoma, the most prevalent malignant bone tumor in children and adolescents, these events dramatically alter the genomic landscape, frequently disrupting key tumor suppressor genes like TP53 and RB1, amplifying oncogene expression, and propelling tumor progression and evolution. This review elucidates how key chromoanagenic mechanisms, such as chromothripsis and chromoanasynthesis, arise from replication stress and impaired DNA repair pathways, ultimately contributing to genomic instability in osteosarcoma. Chromothripsis features prominently in osteosarcoma, occurring in up to 62% of tumor regions and driving intratumoral heterogeneity through persistent genomic crises. Next-generation sequencing, optical genome mapping, and emerging technologies like single-cell sequencing empower researchers to detect and characterize these complex structural variants, demonstrating how a single catastrophic event can profoundly influence osteosarcoma progression over time. While targeted therapies for osteosarcoma have proven elusive, innovative strategies harnessing comprehensive genomic profiling and patient-derived preclinical models hold promise for uncovering tumor-specific vulnerabilities tied to chromoanagenesis. Ultimately, unraveling how these rapid, large-scale rearrangements fuel osteosarcoma’s aggressive nature will not only refine disease classification and prognosis but also pave the way for novel therapeutic approaches to enhance patient outcomes.

Ring chromosomes (RC) are present in 13,000 patients diagnosed with hematological malignancies at the Johns Hopkins University Hospital and identified 98 patients with RCs—90 with myeloid malignancies and 8 with lymphoid malignancies. We also performed a targeted Next-Generation Sequencing (NGS) assay, using a panel of 642 cancer genes, to identify whether these patients harbor relevant pathogenic variants. Cytogenetic analyses revealed that RCs and marker chromosomes of unknown origin are concurrently present in most patients by karyotyping, and 93% of patients with NGS data have complex karyotypes. A total of 72% of these individuals have pathogenic mutations in TP53, most of whom also possess cytogenetic abnormalities resulting in the loss of 17p, including the loss of TP53. All patients with a detected RC and without complex karyotypes also lack TP53 mutations but have pathogenic mutations in TET2. Further, 70% of RCs that map to a known chromosome are detected in individuals without TP53 mutations. Our data suggest that RCs in hematological malignancies may arise through different mechanisms, but ultimately promote widespread chromosomal instability.

Acute promyelocytic leukemia (APL) accounts for approximately 10–15% of newly diagnosed acute myeloid leukemia cases and presents with coagulopathy and bleeding. Prompt diagnosis and treatment are required to minimize early mortality in APL as initiation of all-trans retinoic acid therapy rapidly reverses coagulopathy. The PML::RARA fusion is a hallmark of APL and its rapid identification is essential for rapid initiation of specific treatment to prevent early deaths from coagulopathy and bleeding and optimize patient outcomes. Given limitations and long turnaround time of current gene fusion diagnostic strategies, we have developed a novel amplification-free nanopore sequencing-based approach with low cost, easy setup, and fast turnaround time. We termed the approach CRISPR/Cas9-enriched nanopore sequencing with adaptive sampling (CENAS). Using CENAS, we successfully sequenced breakpoints of typical and atypical PML::RARA fusions in APL patients. Compared with the standard-of-care genetic diagnostic tests, CENAS achieved good concordance in detecting PML::RARA fusions in this study. CENAS allowed for the identification of sequence information of fusion breakpoints involved in typical and atypical PML::RARA fusions and identified additional genes (ANKFN1 and JOSD1) and genomic regions (13q14.13) involving the atypical fusions. To the best of our knowledge, involvements of the ANKFN1 gene, the JOSD1 gene, and the 13q14.13 genomic region flanking with the SIAH3 and ZC3H13 genes have not been reported in the atypical PML::RARA fusions. CENAS has great potential to develop as a point-of-care test enabling immediate, low-cost bedside diagnosis of APL patients with a PML::RARA fusion. Given the early death rate in APL patients still reaches 15%, and ~10% of APL patients are resistant to initial therapy or prone to relapse, further sequencing studies of typical and atypical PML::RARA fusion might shed light on the pathophysiology of the disease and its responsiveness to treatment. Understanding the involvement of additional genes and positional effects related to the PML and RARA genes could shed light on their role in APL and may aid in the development of novel targeted therapies.

Background/objectives: Acute promyelocytic leukemia (APL) is an aggressive subtype of acute myeloid leukemia (AML), characterized by the hallmark translocation t(15;17) resulting in a PML::RARA fusion. Once diagnosed, APL is now considered to be one of the most treatable forms of AML. However, without early detection and treatment, the disease is associated with rapid deterioration and lethal side effects. Methods: We describe a case of diagnostic APL presenting with a normal karyotype, normal RARA break-apart FISH, and unclear, atypical PML/RARA FISH findings. We used optical genome mapping (OGM) to characterize this atypical PML/RARA fusion. Results: OGM allowed for detection of a PML::RARA fusion resulting from a cryptic and complex insertion of PML::RARA into RARA on 17q21.2 whereby a segment of 15q24.1 was inserted into the 17q21.2. The recipient breakpoint of the insertion was at intron 2 of the RARA gene and the donor breakpoint of the insertion was at exon 5/intron 6 of the PML gene. Conclusions: This is the first report of an insertional PML::RARA fusion into the RARA gene on 17q detected by OGM. OGM has demonstrated its utility in a clinical cytogenetics environment, allowing for clearer characterization and diagnosis of various neoplasms.

Background/Objectives: Fluorescence in situ hybridization (FISH) is a standard diagnostic tool for detecting gene fusions and rearrangements in lymphomas but is limited by incomplete genomic coverage, dependence on predefined probes, and difficulty identifying atypical or noncanonical fusion partners. These constraints often result in inconclusive diagnoses in complex lymphoma cases. This study evaluates a novel Hi-C-based sequencing assay from formalin-fixed paraffin-embedded (FFPE) samples to detect clinically significant gene fusions and rearrangements in cases where conventional FISH was inconclusive or expected biomarkers were not detected. Methods: Five diffuse large B-cell lymphoma cases with previously atypical gene fusions or rearrangements by FISH were analyzed using both standard FISH and a Hi-C-based lymphoma assay. Standard FISH was performed using break-apart probes targeting MYC, BCL2, and BCL6, and dual-fusion probes targeting IGH::MYC and IGH::BCL2. The Hi-C assay utilized high-resolution sequencing of FFPE tissue to map chromatin interactions and identify structural variations across the genome and assessment of their clinical relevance. Results: In this series of five lymphoma cases, Hi-C detected additional structural variants beyond those identified by FISH. It identified typical and atypical translocation partners of key oncogenes (MYC, BCL2, BCL6), cryptic breakpoints, and novel genomic events, including TP53 loss, KMT2A amplification, and complex rearrangements, which were undetectable by FISH. The Hi-C assay’s whole-genome coverage enabled comprehensive profiling. Conclusions: The Hi-C-based lymphoma assay offers a transformative diagnostic tool, overcoming FISH limitations by providing unbiased, high-resolution detection of structural variations. This approach enhances diagnostic accuracy and supports personalized therapeutic strategies in lymphoma management, warranting further validation for clinical adoption.